The Application of i-Tasser and related studies to predict the full-length structure, thermodynamic states and functional analysis of the Kirsten Rat Sarcoma Virus (KRAS) protein; An observation of KRAS structural and functional integrity.

The Application of i-Tasser and related studies to predict the full-length structure, thermodynamic states and functional analysis of the Kirsten Rat Sarcoma Virus (KRAS) protein; An observation of KRAS structural and functional integrity. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Application of i-Tasser and related studies to predict the full-length structure, thermodynamic states and functional analysis of the Kirsten Rat Sarcoma Virus (KRAS) protein; An observation of KRAS structural and functional integrity. SDanish Kadir, Tanvir Chowdhury, Mrinal Kranti Saha, Partha Laskar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4784472/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract KRAS (Kirsten Rat Sarcoma Virus) is a Ras (Rat Sarcoma virus) family homologous. Due to its structural characteristics and biochemical properties, it’s been regarded as one of the most common drivers in various cellular signaling pathways. Due to its biochemical and pharmacological importance, KRAS been regarded as the “Holy Grail” of drug discovery. To understand the structure and thermodynamics of this protein, we tried to focus on the confirmational and functional portion of this specific protein. “i-Tesser” is applied to predict the structure of KRAS. A prediction for the secondary and full-length 3D structure been developed. Additional generation of the B factor value, Solvent accessibility, ligand bonded structure, as well as the 5 different thermodynamic state models of the KRAS and their functions are analyzed. The primary, metastable, ligand-bounded reactive states are visualized with the additional application of NAMD (Nanoscale Molecular Dynamics) and VMD (Visualization Molecular Dynamics). The drawn models showcase a C score value ranging between (-0.02 to -1.45) and Cluster density (0.0683 to 0.2835), along with a standard deviation value of (0.704983). Predicted secondary, 3D, and Thermodynamic models are drawn and supported by modeling their Solvent accessibility and B factor values. The 5 different models and the related structural and functional analysis provide additional information about the stability of KRAS structure and its function. Structural Biology KRAS Kirsten Rat Sarcoma Virus i-Tesser Iterative Threading Assembly Refinement C-Score Confidence Score Cluster Density Holy Grail Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 Introduction KRAS, the Kirsten Rat Sarcoma Virus, is a part of the Rat family Sarcoma Virus (RAS). It’s a signal transducer protein. That means it can regulate the various signaling pathways responsible for cellular survival, invasion, growth, and the functional regulation of various channels. KRAS is also a GTPase (Guanosine Triphosphate) activated enzyme-based RAS family oncoprotein. The structure of the KRAS is 85% homologous for the development of different types of Cancer in comparison to other isoforms of the Ras family (i.e.: Harvey Rat Sarcoma Virus, Kirsten Rat Sarcoma Virus and Neuroblastoma Rat Sarcoma Virus). That’s why it’s been regarded as the most significant among all of the RAS family onco-proteins. The bondage of the KRAS at the Receptor Tyrosine Kinase (RTK) or, more specifically, at the Effector Growth Factor Receptor (EGFR) leads to a higher intracellular concentration of the GTP than the GDP (Guanosine di-phosphate), which eventually activates the membrane-bound isopropyl based KRAS protein. Later, this activation of the KRAS stimulates the different cellular pathways such as [RAS- AKT (Adenosine Tyrosine Kinase)-mTOR] responsible for cellular proliferation, which means the growth of the cells, [RAL-Nf-kB] activates the innate immunity, necessary for the regulation of the cellular defense, and [RAF-MEK- ERK] maintains the life cycle of the cells. However, due to various genetic disorders, errors in the synthesis of the KRAS at the site of the ribosome, or the impact of external factors such as the Potential of Hydrogen (pH), temperature, and others, KRAS can go through a series of mutational challenges leading to the development of missense or point mutations. As a result, the Aspartic acid replaces Glycine, making the KRAS hyperactivated. So, whether a receptor bounds at the EGFR or not, KRAS will always remain hyperactivated, eventually impacting the transcription and the transmission to synthesize the myeloma cells or cells with infinite division and growth. Figure 01: The occurrence of the Missense mutation (Glycine by Aspartic acid). This figure illustrates the mechanism leading to a missense mutation or point mutation in the KRAS gene. The primary sequence of the KRAS protein is shown, with a specific focus on the codon sequence where a mutation can occur. The transition from glycine to aspartic acid, highlighted in the sequence, is a common mutation site. External factors such as pressure, temperature, and dysregulation can induce this mutation. The process is depicted as a sequence of events starting from the primary KRAS sequence, influenced by external factors, leading to a missense mutation or point mutation. This mutation results in the substitution of glycine with aspartic acid, significantly impacting the protein's function and contributing to oncogenic transformation. These mutations can lead to the development of various types of Cancer and metabolic disorders. So, to learn about various types of KRAS structures, we must understand their thermodynamic properties first. In the KRAS, the P loop or the Switch-I is the predominant site of mutations. Alongside 7 different mutational states, the application of engineered mutations seems highly frequent (40%). According to some recent analysis, almost 53.5% contain engineered mutations. WT (Wild type), C118S, G12A, G12D, G12V, and Q61H are common. We have performed the study in two completely different ways. At first, we conducted the study, where the first part of the analysis focused on the alterations of the KRAS structure and the development of various disorders. This covers both aspects of the physiological and the functional point of analysis. In the second part of the study, we have drawn the KRAS's Primary (collected from the PDB), Secondary, 3D, and Thermodynamic states using the i-Tesser. Eventually provides us with a complete orientation of the KRAS structure and its functional characteristics. 1.2. Alterations of the KRAS structure and the functions Usually, the mutational transformation results from replacing the Glycine with Aspartic acid, leading to missense mutations or point mutations. [ Note: It’s a type of mutation in which a single amino acid from the residue gets replaced by another amino acid, resulting in various undesired side effects.]. As a result, hyperactivated KRAS is developed, which can function independently to continuously synthesize cellular division and survival. Additionally, the pH of the location gets lower, and it damages various cellular organelles and generates Reactive Oxygen Species (ROS), which is responsible for the occurrence and the resurgence of Cancers and various related disorders. Figure 02: This schematic diagram illustrates the impact of the Rat Sarcoma (RAS) gene and its variants on different cell types and the associated conditions. 1.3. The Role of KRAS in the Development of Cancer As discussed in the previous section, the KRAS mutational characteristics are responsible for tumor cell growth and the development of PDAC (Pancreatic Adenocarcinoma) and CRC (Colorectal Cancer). Metastases of these two types of solid tumors is the reason behind the mortality of thousands worldwide. In this formula, S represents the standard deviation of the population, n is the size of the sample, xxx denotes each value from the given population, and x‾overline{x}x is the mean of the population. In the context of KRAS mutation research, calculating the standard deviation is essential for understanding the variability and dispersion of data points in experimental results, such as gene expression levels or responses to treatments. This statistical tool aids in assessing the reliability and significance of the observed data, thereby contributing to the interpretation of experimental outcomes and the development of targeted therapies. The table gives us an idea about Lung, Pancreatic, Blood, and Colon cancers and their rate of survival in recent times. Figure 03: Bar-chart to highlight the relationship between Cancer types and the Rate of Survival. The bar diagram on the figure visually represents the given information from the provided table with a marginal value of standard deviation. Based upon the upwardly mentioned Table and the graphical representation of the data, the relationship between the different type of cancer and the survival rate in 2018 is resembled and points that even with the application of the various advanced treatments and therapies both Lung and Pancreatic cancer still has the highest morbidity, 95.1%, and 92%, respectively. Application of the standard deviation equation has helped us to figure out the Standard Deviation. KRAS plays a significant role in the occurrence of PDAC, having one of the highest morbidities, making it more interesting due to its sensitive location at the intersection of various blood vessels and with a success rate of 15% only the traditional therapies such as Radiotherapy and Chemotherapy are unable to function appropriately due to the rapid upregulation of alternative compensatory pathways as well as desmoplastic reaction ultimately making it more challenging. To check the mutational and transcriptional landscape of the PDAC, the GTPase KRAS alongside tumor suppressor genes such as p53, CDKN2A (Cyclic-dependent kinase inhibitor type-2), and SMAD4(Mothers Against Decapentologenic-4) exhibited mutations. The mutated KRAS, amplification of their mutant alleles, overexpression of AKT-2, and the activity of the upstream regulator (PI3K) are often elevated in PDAC, increasing the survival of the tumor cells, which can be furtherly defined as a part of the epigenetic dysregulation resulted due to the modifications of the human DNA. Figure 04: The developmental flowchart of Pancreatic Adenocarcinoma (PDAC) The upward figure resembles the development of Pancreatic Adenocarcinoma (PDAC). As soon as the mutation at the KRAS results, it leads to the initiation of the Pancreatic intra-neoplasm stage 1, where the cells on the pancreas go through a series of altered modifications regarded as the initiation of tumorigenesis. The developed hyperplastic lesions are known as pancreatic intraepithelial neoplasia and the intraductal mucinous neoplasms (IPMN) and their prosperity to develop into cancer, which looks like finger (Papillae). Then it comes to stage 2 (mucinous cystic neoplasm) and stage 3 (intraductal papillary mucinous neoplasm) due to the inhibition of cyclic-dependent kinase 2A—eventually, the development of the PDAC results from inhibiting the tumor suppressor gene p53 and SMAD-4. Similarly, the KRAS has also been responsible for the development of Colorectal Cancer. It is considered the most life-threatening among solid tumors and the third most diagnosed cancer in United States of America (USA). According to the latest prediction been made by the American Cancer Society, 153,020 will be diagnosed with cancers, and 52,550 will die in 2023.Similar to Pancreatic Cancer, the initiation of the Colorectal Cancer is also started in the normal epithelial cells. The inhibition of the Adenoma Polypmatous Coli (APC) develops an abnormal growth of the cells called; Adenomatous Polyp, impacted by the missense mutation because of the KRAS. The APC initiates most of both sporadic and hereditary colorectal cancers. The molecular outcome of the APC function loss lies mainly in its inability to downregulate the oncogenic canonical Wnt signaling pathway. The abnormal cascade activation leads to the overexpression of Wnt target genes involved in proliferation. Resulting in the development of lower-grade dysplasia or disordered cellular structures, develops Higher-grade Dysplasia at the inhibition of the DCC gene [ Note: both cellular changes can easily be observed in the figure entitled “The physiological development of Colorectal cancer”], a putative tumor suppressor gene. The somatic mutations of the DCC gene and the netrin-1 during the organization of the spinal cord led to the position of DCC inactivation, significantly impacting tumorigenesis. To eventually inhibit the p53 gene, resulting in Colorectal Cancer. Figure 05: The developmental flowchart of Colorectal Cancer (CRC) Figure 06: The KRAS regulated various cellular pathways. 1.4. The Role of KRAS in the Development of Rasopathies The hyperactivation of the KRAS can also lead to the development of Rasopathies. It’s a series of rare disorders where the alterations in the “Ras (Rat Sarcoma Virus)/MAPK (Mitogen Associated Phosphate Kinase)” signaling pathways occur. A table is given below to mention various KRAS dependent Rasopathies, their symptoms, and causes. Table 02: KRAS-dependent Rasopathies, Symptoms, and their causes Rasopathies Symptoms Cause Neurofibromatosis type 1 Neurofibromas, problems with the eyes and the bones NF-1 gene mutation Noonan Syndrome Short stature, heart defects, unusual facial features. Altered KRAS, RAF-1 gene Cardio-facio-cutaneous Syndrome Small chin and short nose Altered KRAS, BRAF, MEK1/MEK2 1.5. The Role of the KRAS in the Development of Diabetes Mellitus Type-2 In addition to Cancer and Rasopathies, the KRAS can also play a significant role in the development of Diabetes Mellitus Type-2. The bondage between the advanced glycolytic endpoints at the receptor activates the Phosphate Kinase C(PKC), eventually impacting the secretions of the NADPH oxidase. This increases the intercellular concentration of the Reactive Oxygen Species (ROS) and damages the DNA. All these results are due to the mutations in the KRAS and affect the secretion of the Cytochrome P450 and the Glycolysis process by inhibiting the Hexosamine kinase and Pentose Phosphate pathways. These alterations of the cascade develop insulin-resistant diabetes mellitus type-2, functioning in an unusual positive feedback mechanism rather than the natural positive feedback mechanism. A drop in the glucagon level in the blood leads to the rise of insulin concentrations in the peripheral tissues. This is an example of the negative feedback mechanism. However, in the positive feedback process, they work in an agonistic pattern by increasing each other’s volume, which seems to be synchronizing. That’s how the insulin-resistant diabetes develops. Figure 07: The developmental process of the diabetes mellitus type-2, due to the mutation of the KRAS. The figure illustrates the pathway involved in the development of type-2 diabetes mellitus as a consequence of KRAS mutation. The process begins with Advanced Glycation End-products (AGE) interacting with the Receptor for Advanced Glycation End-products (RAGE), leading to the activation of Protein Kinase C (PKC). This activation stimulates NADPH oxidase, resulting in the production of Reactive Oxygen Species (ROS). The ROS, in turn, influences various metabolic processes, including the activity of cytochrome P450 and the regulation of glucose levels. ROS also impacts the pentose phosphate pathway and hexosamine kinase activity within the inner membrane, both of which are linked to glycolysis. The interconnected pathways highlight the complex biochemical cascade triggered by KRAS mutation, contributing to the pathogenesis of type-2 diabetes mellitus. Methodology Figure 08: The Primary Amino acid Sequence of the KRAS (Kirsten Rat Sarcoma Virus) The collected primary sequence of the Kirsten Rat Sarcoma Virus (KRAS) from the Protein Data Bank (PDB) [ Note: PDB ID: 7X8N] is being furtherly analyzed by the application of the Bioinformatic tool “i-Tesser; Iterative threading Assembly Refinement.” According to the critical assessment of techniques for protein structure prediction (CASP), the i-Tesser was awarded the first position for the CASP-7 and CASP-8. That’s the reason behind the choice of the i-Tesser. It focuses on the principles of the Monte Carlo Simulation technique for 3D visualization. It utilizes a combination between the “Nanoscale Molecular Dynamics (NAMD)” and “Visualization Molecular Dynamics (VMD).” The primary sequences of the protein get through the “Local meta-threading server (LOMETS).” to develop a template for the fragments. Later, the templates are assembled based on their increasing decoy-based optimization potential value. The structure is usually clustered with the molecules with the lowest energy level developed by the Cluster centroid with the “SPICKER” software application. The same set of molecules is then reassembled, and from there, the molecules with the lowest energy structure show the optimization values of the H-bond to generate a finalized model. During this phase of 3Dstructural visualization, both the “Nanoscale Molecular Dynamics (NAMD)” and the “Visualization Molecular Dynamics (VMD)” are applied. This helps to simulate larger-size biomolecules ranging from 10,000 to 10,000,000 atoms, and the various rendering styles and methods differentiate between regions. Figure 09: The ligand-bound structure of the KRAS (Kirsten Rat Sarcoma Virus). The figure illustrates the three-dimensional structure of the KRAS protein in its ligand-bound state. The protein backbone is depicted in green, highlighting the intricate folding and secondary structural elements of the molecule. The bound ligand, visible within the structure, underscores key interaction sites critical for KRAS function and its role in signalling pathways. This structural representation provides valuable insights into the molecular interactions and conformational changes associated with ligand binding, aiding in the understanding of KRAS-related mechanisms and potential therapeutic targeting. In this way, the top 5 structural models and the ligand-bounded structures are estimated based on their C score values using the algorithmic ResQ analysis, indicating a model of global topology. Moreover, the i-Tesser helps to predict a comparatively stable form of the secondary structures. This has been further supported by the higher density of the amino acid molecules at the 2 nd quadrant of the Ramachandran plot. Figure 10: The Ramachandran Plot of the KRAS (Kirsten Rat Sarcoma Virus). The figure displays the Ramachandran plot for the KRAS protein structure, illustrating the distribution of phi (Φ) and psi (Ψ) dihedral angles in the polypeptide chain. Each point on the plot represents a residue in the protein structure. The plot is divided into regions indicating the preferred conformations of the polypeptide backbone. The yellow areas denote allowed regions, while the red areas signify the most favoured regions. A notable feature in this plot is the left-handed beta-sheet structure, highlighted with a yellow arrow pointing towards the cluster of data points in the upper left quadrant, corresponding to the left-handed beta-sheet conformation. This plot provides insights into the conformational constraints and flexibility of the KRAS protein, crucial for understanding its functional dynamics. The B factor structure value of the KRAS and the solvent accessibility score are more detail-oriented values to explain the thermodynamic properties of the structural stability and the functional ability of the KRAS protein, which has been analyzed and drawn for the sake of the multiple angle analysis. In addition, we have utilized “ Microsoft Excel-2022 ” to analyze the statistical relationship between the “ C-Score and Cluster Density .” and “the extent of comparative mortality between various forms of cancers.” Result The predicted secondary structure of the KRAS and their various structural forms are drawn based on their confidence score values, ranging from ( 0-9 ). The higher the means, the more confident the prediction of the structure. Figure 10: KRAS confidence score values Similarly, the B factor structure values are estimated, referring to the thermal stability score having a range of (-2,5). The lower the value, the more stable the structures. So, the more negative the value more stable it is. Figure 11: The B factor value of the KRAS (Kirsten Rat Sarcoma Virus) Additionally, the solvent accessibility score is estimated to depict the reactive aspect of the sequence. These values range between 0(buried residue) and 9(highly exposed residue). To analyze the relationship extent between 5 different models of the KRAS, both the C score and the Cluster density values are analyzed. Where the C-Score value lingers around (-0.02) to (-1.45), and the cluster density varies from (0.0683) to (0.2835), with a Standard Deviation (SD) value of (0.704983). ANOVA: Single Factor (Confidence Score) SUMMARY Groups Count Sum Average Variance 1 4 14 3.5 1.666667 -0.02 4 -4.92 -1.23 0.055533 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 44.7458 1 44.7458 51.96354 0.000361 5.987378 Within Groups 5.1666 6 0.8611 Total 49.9124 7 ANOVA: Single Factor (Cluster Density) SUMMARY Groups Count Sum Average Variance 1 4 14 3.5 1.666667 0.2835 4 0.3468 0.0867 0.000439 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 23.30123 1 23.30123 27.95412 0.001852 5.987378 Within Groups 5.001317 6 0.833553 Total 28.30255 7 Both ANOVA analyses (for C-scores and Cluster Density) indicate statistically significant differences between the groups. The F-values in both cases are much larger than the F critical values, and the P-values are significantly lower than 0.05. This suggests that the structural models of KRAS have significantly different C-scores and Cluster Density values. Figure 12: KRAS Structural Stability. This bar graph illustrates the relationship between five different structural models of the KRAS protein, C-scores, and Cluster Densities. The C-scores are represented by red bars, while the Cluster Densities are represented by black bars. The graph also includes trend lines for both C-scores (dotted blue line) and Cluster Densities (dotted orange line). Model 01 :C-score: -0.02, Cluster Density: 0.2835, Model 02 : C-score: -1.18, Cluster Density: 0.0895, Model 03 : C-score: -1.37, Cluster Density: 0.0739, Model 04 : C-score: -0.92, Cluster Density: 0.1151, Model 05 : C-score: -1.45, Cluster Density: 0.0683, Standard Deviation (SD) : C-score: 0.704983 (represented as a red bar for a standard deviation for C-scores). The graph shows that Model 01 has the highest Cluster Density and the least negative C-score, indicating potentially better structural stability compared to the other models. Model 05 has the lowest C-score and Cluster Density, suggesting it might be the least stable. The trend lines provide a visual representation of the overall trends in C-scores and Cluster Densities across the models. This graph provides insight into the structural stability of different KRAS protein models, with implications for their reliability in research and potential therapeutic applications. Higher Cluster Density and less negative C-scores are indicative of more reliable structural models. Discussion The KRAS is a 189 amino acid-long protein substance. The molecular weight of the KRAS is 21KDa, which has no structural sub-unit. It has 5 alpha helices and 6 beta-strand; it’s one of the three isoforms of the RAS family protein. Been divided into two major regions—G domain (1-166) amino acid chain and Hypervariable region (HVR). lasts from (167-189) amino acids. The KRAS's generalized primary amino acid sequence has a specialized structural motif called; the P loop. This is also popularized as the “Walker A motif.”, the structural motif is highlighted by the green color. Where we can observe the existence of the primary amino acid sequence, a pattern of “ GAAAAGKS ,” Where the A stands for any amino acid molecule. Figure 13: The 3D structure of the KRAS (Kirsten Rat Sarcoma Virus) Uusally, the P-loop is a structural pocket where the proteins can easily bind. The G domain can be furtherly subdivided into 3 different parts; P-loop (10-17), Switch-I (30-40), and Switch-II (59-76). Similar to the other related mutational hotspots, such as; Switch-I and Switch-II, are highlighted with red and purple colors respectively. As for the C terminal-based Hypervariable region, it’s been showcased with a navy blue color. The KRAS has 2 isoforms [ Note: A chemically active compound generated from the same prtoein] KRAS4A and KRAS4B, differentiated at the following sites; 151,153,165,166 and the Hypervariable regions. For the necessity the HVR, lately can go through the following post-translational modifications; Farnesylation, Methylation, and Palmitoylation. Insertion of the primary amino acid sequence in the i-Tesser resulted in the prediction of the secondary protein structure. The higher the confidence score, the more reliable the predicted structure. A score of 9 meaning the secondary structure of the specific amino acid of the KRAS is more stable and realible. The Helix, Strand, and Coil structures are showcased with different colors. Which indicates that the predicted coiled from of the structure seems to be structurally more stable than the Helix and Strand conformations. The Ramachandran plot supports the secondary structure of the protein, defining the torsion angle between the “psi” and “phi.” bond between the Carbon atoms. To make an accurately predicted protein structure, here, the PDBsum server is used to generate the Ramachandran plot. Where the greater density of the amino acids at the 2nd quadrant of the Ramachandran plot denotes the beta-stranded left-handed protein conformation. Finally, with the application of the NAMD and VMD principles, the i-Tesser will help us to draw the 3D structure of the KRAS. An ideal 3D structure of the KRAS is shown in the image. Where the pink color depicts the alpha-helices as a twisted structure. Whereas, the beta-strand is highlighted with the yellow color. The end of the C terminus is the White-colored one and just under the C terminus, it’s the Hypervariable region. To check the thermal mobility of the KRAS protein, a frequency curve been drawn to provide the details about the structural stability of the amino acid residues. Normalized B factor value for a target protein is predicted by applying template-based assignment and profile-based prediction. The residues with higher than “0” B-factor values are comparatively less stable in experimental conditions. So, the negative values of the amino acid residues mean a comparatively more stable structure. The strand structures of the amino acid residues highlighted with green having a lower value, so they are structurally more stable. The helix and coil structures highlighted with purple and black colors are structurally either helix or coiled-shaped. To furtherly highlight the thermodynamic and structural characteristics of the predicted structure, we have performed the solvent accessibility score analysis. Figure 14: The solvent accessibility score of the KRAS (Kirsten Rat Sarcoma Virus) The figure presents the solvent accessibility score of the KRAS protein sequence. The sequence is displayed along the top, ranging from residues 1 to 180. Below the sequence, the predicted solvent accessibility scores are provided, where values range from 0 to 9. A score of 0 indicates a buried residue, while 9 signifies a highly exposed residue. This visual representation helps in understanding the exposure level of each residue in the KRAS protein, which is crucial for insights into its structure and interaction with other molecules. Figure 15: Normal Probability Plot. This figure displays a Normal Probability Plot of the predicted values against their sample percentiles. The predicted values follow a normal distribution. The horizontal axis represents the sample percentiles, while the vertical axis represents the predicted values. Data Points (Series 1): The blue diamonds represent the observed predicted values plotted against their corresponding sample percentiles. Trend Line (Linear Series1): The black line represents the best-fit line for the data points. The equation of the line is y=0.0236xy = 0.0236xy=0.0236x and the coefficient of determination (R²) is 0.6028, indicating that approximately 60.28% of the variance in the predicted values can be explained by the sample percentiles. Statistical Analysis Regression Statistics Multiple R 0.354578 R Square 0.125726 Adjusted R Square 0.120786 Standard Error 1.405166 Observations 179 ANOVA df SS MS F Significance F Regression 1 50.25799 50.25799 25.45363 1.12E-06 Residual 177 349.485 1.974492 Total 178 399.743 Coefficients Standard Error t Stat P-value Lower 95% Upper 95% Lower 95.0% Upper 95.0% Intercept 1.849373 0.212702 8.694652 2.31E-15 1.429614 2.269132 1.429614 2.269132 1 -0.01025 0.002033 -5.04516 1.12E-06 -0.01427 -0.00624 -0.01427 -0.00624 The lower score means less exposure, and the higher score means relatively more exposed regions. The greater the exposure, the larger the surface volume ratio, making it more suitable to drug-protein conjugation. As for the drug-protein conjugation, the overall structure of the KRAS is shown with the green color and the bounded molecules it’s been highlighted with different other colors. The conjugation always depicts the thermodynamic states of the KRAS’s 3D structural models. Figure 16: The thermodynamic states of the KRAS. The images are varied based on the values of the C score. The C Score values. (a) -0.02, (b) -1.18, (c) -1.37, (d) -0.92, and (e) -1.45. Here, upwardly predicted five different models of the KRAS are drawn based on the C score value of the KRAS. Where (a) has a C score value of (-0.02) and is relatively more stable than model (e), which has a C score value of (-1.45). As for the model (b), (c), and (d), they have different C score values, respectively. The C-score value of model(b) is (-1.18), model (c) is (-1.37), and model (d) is (-.92). These models are very important to analyze the mutational status of the KRAS. Among the primary amino acid sequences, the G12S, G13C, Q61R, Q61K, and Q61X are very important for their mutational states. The different mutant features of the KRAS are shown in the image (p), and each of them is structurally different from the other, so as their specific actions. 4. Limitations of this study and the related future research opportunities Although the authors have given different thermodynamic states of the KRAS or the mutant states of the KRAS, they are still required to provide the structural point of view of all 7 different mutant states of the KRAS protein. Also, the confidence score value among the amino acid residue is unequal, and this score usually varies. That indicates a level of uncertainty among the predicted structures. Additionally, the dynamics of the KRAS's dimeric and oligomeric complexes are furtherly needed to be researched. So, to resolve these challenges, state art models such as the Markov State Model (MSM) with a combination of the Cryo-EM (based on the principles of the NAMD and VMD) must be implemented. Conclusion In conclusion, As a gene, KRAS plays a central role in the initiation, invasion, treatment, resistance, and reoccurrence of Colorectal and Pancreatic Cancers. A detailed idea about the KRAS and its functional and structural overview deciphers the opportunity to focus on this “Undruggable Oncoprotein”. So to investigate thoroughly about the KRAS, we have tried to focus on its conformational interest. This is necessary to analyze in the upcoming times to resolve the full-length structural and thermodynamic challenges associated with the KRAS. Declarations Competing Interest: The author declared that he has no known competing financial or personal relationships that could have appeared to influence the work reported in this research dissertation. Ethics approval and consent to participate Consent for publication: All the authors mutually agreeing for the publication this this study Availability of data and materials Funding: Not applicable Authors' contributions: the generation of the data to the interpretation and ultimately the development of the manuscript, each part of the complete manuscript been developed by DK under the guidance of MK (Mrinal Kranti Saha), Where the rest of the authors TC (Tanvir Chowdhury) have contributed especially to the screening and image visualization. Acknowledgments The author would like to thank the University of Texas Rio Grande Valley, I-Tesser tool, and its developer Dr. Zhang et.al. from University of Michigan, Ann Arbor. We would furtherly like to share our gratitude towards our family members and well-wishers for their continuous support in developing this precious research manuscript and the author DK wants to share his special thanks to First Lutheran Church, Brookings, South Dakota-57006 for their hospitality. References Al Qahtani WS (2020) Utility of KRAS Gene and Clinicopathological Features in the Assessment of the Risk of Type 2 Diabetes in the Etiology of Colon Cancer. Global Medical Genetics Buscial L (2020) Role of oncogenic KRAS in diagnosing, prognosis, and treating pancreatic cancer. Nature Reviews Gastroenterology and Hepatology Cancer, Facts, Figures (2018) American Cancer Society C.Phillips J (2020) Scalable molecular dynamics on CPU and GPU architectures with NAMD. J Clin Phys, 153 D., S. (2017) LiteMol suite: Interactive web-based visualization of large-scale macromolecular structure data. Nature Methods Husic E, B (2018) Markov State Models: From an Art to a Science. Journal of American Chemical Society Ferrer I (2018) KRAS-Mutant non-small cell lung cancer: From biology to therapy. Lung Cancer Neel F, N (2011) The RalGEF-Ral Effector Signaling Network: The Road Less Traveled for Anti-Ras Drug Discovery. Gene Cancer, 3(2) Frenknel D (2002) Understanding Molecular Simulation, vol 2. Elsevier Garzilli I (2018) Design principles of the paradoxical feedback between pancreatic alpha and beta cells. Sci Rep, 8 Hillig Christian Roman (2022) Targeting RAS oncogenesis with SOS1 inhibitors. Advances in Cancer Research, vol 153. Elsevier Huang L (2021) KRAS mutation: From undruggable to druggable in cancer. Signal Transduction and Targeted Therapy K.Shimanshu D (2017) RAS Proteins and Their Regulators in Human Disease. Cell, p 170 Friedman M (2022) J. Neurofibromatosis 1. Gene Reviews Noonan syndrome (2023) National Center for Advancing Translational Sciences Nyíri K (2020) Structure-based inhibitor design of mutant RAS proteins—A paradigm shift. Cancer Metastasis Rev, 39 Orth M (2019) Pancreatic ductal adenocarcinoma: Biological hallmarks, current status, and future perspectives of combined modality treatment approaches. Radiat Oncol, 14 Panstar T (2020) The current understanding of KRAS protein structure and dynamics. Comput Struct Biotechnol, 18 Prakash P (2013) Lessons from computer simulations of Ras proteins in solution and in membrane. Biochimica et Biophysica Acta (BBA) - General Subjects, 1830 Protein Data Bank (2023) Rebecca L, Siegel MPH, PhD NSWMMHA, Andrea Cercek MD, Robert A Smith PhD, Ahmedin Jemal DVM, PhD. (2023). Colorectal cancer statistics, 2023. A Journal Cancer for the Clinicians Roberts A (2006) The cardiofaciocutaneous syndrome. J Med Genet, 4 Punekar R, S (2022) The current state of the art and future trends in RAS-targeted cancer therapies. Nat Reviews Clin Oncol, 19 Shibata H (2017) Mucinous Cystic Neoplasms Lined by Abundant Mucinous Epithelium Frequently Involve KRAS Mutations and Malignant Progression. Anticancer Research Simon K (2016) Colorectal cancer development and advances in screening. Clin Invest Aging, 11 Stephanie PM (2018) RAS variant signaling. Biochem Soc Trans, 46(5) T.Ihle N (2012) Effect of KRAS Oncogene Substitutions on Protein Behavior: Implications for Signaling and Clinical Outcome. Journal of the National Cancer Institute Wang K-B (2022) Structural insight into the bulge-containing KRAS oncogene promoter G-quadruplex bound to berberine and coptisine. Nature Communications Wiltgen M (2019) Algorithms for Structure Comparison and Analysis: Homology Modelling of Proteins. Encyclopedia of Bioinformatics and Computational Biology, 1 Yang J, Yang J, Zhang Y (2015) I-TASSER server: New development for protein structure and function predictions. Nucleic Acids Research, 43: W174-W181 (2015). Nucleic Acid Research Zhang M (2019) The structural basis for Ras activation of PI3Kα lipid kinase. Physical Chemistry Physical Physics Zheng W (2021) Folding non-homologous proteins by coupling deep-learning contact maps with I-TASSER assembly simulations. Cell Reports Methods Additional Declarations The authors declare no competing interests. Supplementary Files Table01KRAS.docx Table02KRAS.docx Table03KRAS.docx GraphicalAbstract.docx Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4784472","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":333857133,"identity":"0b2f563e-594a-47fd-9914-82bdd158ce1c","order_by":0,"name":"SDanish Kadir","email":"","orcid":"https://orcid.org/0000-0003-0708-2232","institution":"University of Texas Rio Grande Valley","correspondingAuthor":false,"prefix":"","firstName":"SDanish","middleName":"","lastName":"Kadir","suffix":""},{"id":333857134,"identity":"fbe33518-dbc9-434c-ac55-f2c8cf12aa7c","order_by":1,"name":"Tanvir Chowdhury","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIiWNgGAWjYLCCBDBiYGD8YGADohoP4FPNg6yFWaIgDaSlgbAWBqgWBp4Ph8E0Xi327GePSTzccyePf/bpxA8SBuft1rYfBtpSYxON0xaevDSJhGfPiiXO5W6WKDC4nbztTCJQy7G03AacDssxNkg4cDix4QzvBgkJoBazA0AtjA2HcWvhfwPRMv8M7+YfPAbnks3OPySgRSLH8AFIy4YzvNskeAwO2JndIGTLjTcgLc8SNwK1WEsYJCeY3QDakoDHL+z9OQYHfxy4kzgP6LCbH/7Y2ZudT3/44EONDU4tUHAAzkoEq0zArxxViz1hxaNgFIyCUTDSAADYv2pmaM05AgAAAABJRU5ErkJggg==","orcid":"","institution":"University of Texas Rio Grande Valley","correspondingAuthor":true,"prefix":"","firstName":"Tanvir","middleName":"","lastName":"Chowdhury","suffix":""},{"id":333857135,"identity":"770a4741-cb52-4ab8-ba39-fb77320f0a1c","order_by":2,"name":"Mrinal Kranti Saha","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYFACxgYGhgIGBglm5oMPgFwePuK0GAC1sLclG4C0sBFnE0gLzxkzCRCboBbd2YfbHvwwqLOXnJFgVvk1x06GjYH54aMbeLSYnUtsN+wxOJw4WyIh7bbstmSgw9iMjXPwaTnD2CbBY3AgQU4i4dhtyW3MQC08bNKEtEj+ATpMTiKxrVhyWz1xWqR5DJgZZ/McZmP8uO0wkVpkgH6Z2d7GLM247TgPGzNBv7A/k3xTUWcvcZj/48ef26rt+dmbHz7GpwUFMPOASWKVgwDjD1JUj4JRMApGwYgBAFn7QkQUC9kWAAAAAElFTkSuQmCC","orcid":"","institution":"University of Texas Rio Grande Valley","correspondingAuthor":true,"prefix":"","firstName":"Mrinal","middleName":"Kranti","lastName":"Saha","suffix":""},{"id":387536307,"identity":"a50a915c-8009-44d1-a78a-360e5c259754","order_by":3,"name":"Partha Laskar","email":"","orcid":"","institution":"University of Texas Rio Grande Valley","correspondingAuthor":false,"prefix":"","firstName":"Partha","middleName":"","lastName":"Laskar","suffix":""}],"badges":[],"createdAt":"2024-07-22 22:06:20","currentVersionCode":2,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-4784472/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-4784472/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71691227,"identity":"786c4866-519e-4723-9826-3b7a7f7ec787","added_by":"auto","created_at":"2024-12-17 18:16:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":179746,"visible":true,"origin":"","legend":"\u003cp\u003eThe occurrence of the Missense mutation (Glycine by Aspartic acid).\u003c/p\u003e\n\u003cp\u003eThis figure illustrates the mechanism leading to a missense mutation or point mutation in the KRAS gene. The primary sequence of the KRAS protein is shown, with a specific focus on the codon sequence where a mutation can occur. The transition from glycine to aspartic acid, highlighted in the sequence, is a common mutation site. External factors such as pressure, temperature, and dysregulation can induce this mutation. The process is depicted as a sequence of events starting from the primary KRAS sequence, influenced by external factors, leading to a missense mutation or point mutation. This mutation results in the substitution of glycine with aspartic acid, significantly impacting the protein's function and contributing to oncogenic transformation.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4784472/v2/9bdc63a829f713abe975a4b3.png"},{"id":71691229,"identity":"6923f1e4-b635-4608-8b8a-a04df5145417","added_by":"auto","created_at":"2024-12-17 18:16:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":404879,"visible":true,"origin":"","legend":"\u003cp\u003eThis schematic diagram illustrates the impact of the Rat Sarcoma (RAS) gene and its variants on different cell types and the associated conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTop Row\u003c/strong\u003e \u003cstrong\u003eRAS Gene (Red Cell)\u003c/strong\u003eRepresents the RAS gene, \u003cstrong\u003eDiabetic Cell (Blue Cell)\u003c/strong\u003e Indicates a cell affected by diabetes, \u003cstrong\u003eCancer Cell (Light Pink Cell)\u003c/strong\u003eDenotes a cancerous cell, \u003cstrong\u003eRasopathies (Light Green Cell)\u003c/strong\u003eRefers to a group of disorders caused by mutations in genes of the \u003cstrong\u003eRAS/MAPK\u003c/strong\u003epathway. \u003cstrong\u003eMiddle Section\u003c/strong\u003e \u003cstrong\u003eRat Sarcoma Virus\u003c/strong\u003e The overarching term for the RAS gene variants, \u003cstrong\u003eKirsten Rat Sarcoma Virus\u003c/strong\u003eShown with a green mutation on the RAS gene, \u003cstrong\u003eN-Rat Sarcoma Virus\u003c/strong\u003eDepicted with a brown mutation, \u003cstrong\u003eH-Rat Sarcoma Virus\u003c/strong\u003e Displayed with a blue mutation. \u003cstrong\u003eRight Section\u003c/strong\u003e \u003cstrong\u003eDiabetic Cell\u003c/strong\u003e The blue cell represents diabetic conditions, \u003cstrong\u003eCancer Cell\u003c/strong\u003e The red cell shows a progression or effect on cancer cells, \u003cstrong\u003eRasopathies\u003c/strong\u003eVisual representation of the disorders in the \u003cstrong\u003eRAS/MAPK\u003c/strong\u003e pathway, depicted with a diagram showing cellular interactions and mutations.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4784472/v2/74b384f65545e8ea7c609a93.png"},{"id":71691223,"identity":"446b9378-7c4a-4606-88d6-143c096f85ec","added_by":"auto","created_at":"2024-12-17 18:16:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":32023,"visible":true,"origin":"","legend":"\u003cp\u003eBar-chart to highlight the relationship between Cancer types and the Rate of Survival.\u003c/p\u003e\n\u003cp\u003eThe bar diagram on the figure visually represents the given information from the provided table with a marginal value of standard deviation. Based upon the upwardly mentioned Table and the graphical representation of the data, the relationship between the different type of cancer and the survival rate in 2018 is resembled and points that even with the application of the various advanced treatments and therapies both Lung and Pancreatic cancer still has the highest morbidity, 95.1%, and 92%, respectively. Application of the standard deviation equation has helped us to figure out the Standard Deviation. KRAS plays a significant role in the occurrence of PDAC, having one of the highest morbidities, making it more interesting due to its sensitive location at the intersection of various blood vessels and with a success rate of 15% only the traditional therapies such as Radiotherapy and Chemotherapy are unable to function appropriately due to the rapid upregulation of alternative compensatory pathways as well as desmoplastic reaction ultimately making it more challenging.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4784472/v2/de512c1c64fd3357fbcdd93b.png"},{"id":71691233,"identity":"85d47528-90d9-451e-b0e3-15dc4dee4416","added_by":"auto","created_at":"2024-12-17 18:16:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":66529,"visible":true,"origin":"","legend":"\u003cp\u003eThe developmental flowchart of Pancreatic Adenocarcinoma (PDAC)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4784472/v2/c5c47f4e3a892d3610ea042b.png"},{"id":71691228,"identity":"c7c3b03b-259d-496c-8317-617067a0bad5","added_by":"auto","created_at":"2024-12-17 18:16:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":98502,"visible":true,"origin":"","legend":"\u003cp\u003eThe developmental flowchart of Colorectal Cancer (CRC)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4784472/v2/9b43de8ae985ecf46632f667.png"},{"id":71692250,"identity":"08118f13-8b8d-4c83-b754-d7a99a190382","added_by":"auto","created_at":"2024-12-17 18:24:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":822251,"visible":true,"origin":"","legend":"\u003cp\u003eThe KRAS regulated various cellular pathways.\u003c/p\u003e\n\u003cp\u003e(a) The (\u003cstrong\u003eP13K-AKT-mTOR\u003c/strong\u003e) is responsible for cellular proliferation. (b) (\u003cstrong\u003eRAF-MEK-ERK\u003c/strong\u003e) regulates the cellular survival, (c) (\u003cstrong\u003eRAL-NF-kB\u003c/strong\u003e) leads to the activation of the cellular immunity. This figure illustrates the KRAS signalling pathways involved in cell survival and proliferation. Activation of EGFR (Epidermal Growth Factor Receptor) leads to KRAS transitioning from its inactive GDP-bound state to the active GTP-bound state. KRAS-GTP activates the RAF/MEK/ERK pathway, promoting cell survival, and the PI3K/AKT/mTOR pathway, driving cell proliferation. Key components and interactions, including EGFR, KRAS-GDP, KRAS-GTP, RAF, MEK, ERK, PI3K, AKT, and mTOR, are shown. Dysregulation of these pathways due to KRAS mutations contributes to oncogenesis, highlighting potential therapeutic targets in cancer treatment.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4784472/v2/312a8ee176eff4141dabda10.png"},{"id":71691234,"identity":"55767e56-c3ef-4251-8a18-a5f3aa5bbb80","added_by":"auto","created_at":"2024-12-17 18:16:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":114932,"visible":true,"origin":"","legend":"\u003cp\u003eThe developmental process of the diabetes mellitus type-2, due to the mutation of the KRAS.\u003c/p\u003e\n\u003cp\u003eThe figure illustrates the pathway involved in the development of type-2 diabetes mellitus as a consequence of KRAS mutation. The process begins with Advanced Glycation End-products (AGE) interacting with the Receptor for Advanced Glycation End-products (RAGE), leading to the activation of Protein Kinase C (PKC). This activation stimulates NADPH oxidase, resulting in the production of Reactive Oxygen Species (ROS). The ROS, in turn, influences various metabolic processes, including the activity of cytochrome P450 and the regulation of glucose levels. ROS also impacts the pentose phosphate pathway and hexosamine kinase activity within the inner membrane, both of which are linked to glycolysis. The interconnected pathways highlight the complex biochemical cascade triggered by KRAS mutation, contributing to the pathogenesis of type-2 diabetes mellitus.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4784472/v2/9a4d3dfae78b820ec5b77986.png"},{"id":71691231,"identity":"e3372cbf-09b7-4c9f-8b3e-4fa9de10d9f0","added_by":"auto","created_at":"2024-12-17 18:16:47","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":194318,"visible":true,"origin":"","legend":"\u003cp\u003eThe Primary Amino acid Sequence of the KRAS (Kirsten Rat Sarcoma Virus)\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4784472/v2/6832f31afb18f050fd8be41b.png"},{"id":71691243,"identity":"c335e619-b41d-4a47-b3ba-4aa910a24ff2","added_by":"auto","created_at":"2024-12-17 18:16:49","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":369993,"visible":true,"origin":"","legend":"\u003cp\u003eThe ligand-bound structure of the KRAS (Kirsten Rat Sarcoma Virus).\u003c/p\u003e\n\u003cp\u003eThe figure illustrates the three-dimensional structure of the KRAS protein in its ligand-bound state. The protein backbone is depicted in green, highlighting the intricate folding and secondary structural elements of the molecule. The bound ligand, visible within the structure, underscores key interaction sites critical for KRAS function and its role in signalling pathways. This structural representation provides valuable insights into the molecular interactions and conformational changes associated with ligand binding, aiding in the understanding of KRAS-related mechanisms and potential therapeutic targeting.\u003c/p\u003e","description":"","filename":"90.png","url":"https://assets-eu.researchsquare.com/files/rs-4784472/v2/245ad6ad05e0717aa4e3a5ec.png"},{"id":71692252,"identity":"450e8df8-070f-4fbe-8c61-cb54eb4e48ec","added_by":"auto","created_at":"2024-12-17 18:24:48","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":294547,"visible":true,"origin":"","legend":"\u003cp\u003eThe Ramachandran Plot of the KRAS (Kirsten Rat Sarcoma Virus).\u003c/p\u003e\n\u003cp\u003eThe figure displays the Ramachandran plot for the KRAS protein structure, illustrating the distribution of phi (Φ) and psi (Ψ) dihedral angles in the polypeptide chain. Each point on the plot represents a residue in the protein structure. The plot is divided into regions indicating the preferred conformations of the polypeptide backbone. The yellow areas denote allowed regions, while the red areas signify the most favoured regions. A notable feature in this plot is the left-handed beta-sheet structure, highlighted with a yellow arrow pointing towards the cluster of data points in the upper left quadrant, corresponding to the left-handed beta-sheet conformation. This plot provides insights into the conformational constraints and flexibility of the KRAS protein, crucial for understanding its functional dynamics.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4784472/v2/fd99be5a563a5314408b9618.png"},{"id":71691241,"identity":"7a98b669-4273-4395-a11c-0599ed2f40d1","added_by":"auto","created_at":"2024-12-17 18:16:49","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":327748,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 10:\u003c/strong\u003e KRAS confidence score values\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-4784472/v2/209ef75321216e17ec05cefb.png"},{"id":71691232,"identity":"49b53d3c-5c28-4860-a28c-2da57201154d","added_by":"auto","created_at":"2024-12-17 18:16:48","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":188058,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 11: \u003c/strong\u003eThe B factor value of the KRAS (Kirsten Rat Sarcoma Virus)\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-4784472/v2/8851e9f7b7be794f12ca8040.png"},{"id":71691225,"identity":"e40e535d-d0db-455b-bd69-252e83dbedd6","added_by":"auto","created_at":"2024-12-17 18:16:47","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":32131,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 12: \u003c/strong\u003eKRAS Structural Stability.\u003c/p\u003e\n\u003cp\u003eThis bar graph illustrates the relationship between five different structural models of the KRAS protein, C-scores, and Cluster Densities. The C-scores are represented by red bars, while the Cluster Densities are represented by black bars. The graph also includes trend lines for both C-scores (dotted blue line) and Cluster Densities (dotted orange line). \u003cstrong\u003eModel 01\u003c/strong\u003e:C-score: -0.02, Cluster Density: 0.2835, \u003cstrong\u003eModel 02\u003c/strong\u003e: C-score: -1.18, Cluster Density: 0.0895, \u003cstrong\u003eModel 03\u003c/strong\u003e: C-score: -1.37, Cluster Density: 0.0739, \u003cstrong\u003eModel 04\u003c/strong\u003e: C-score: -0.92, Cluster Density: 0.1151, \u003cstrong\u003eModel 05\u003c/strong\u003e: C-score: -1.45, Cluster Density: 0.0683, \u003cstrong\u003eStandard Deviation (SD)\u003c/strong\u003e: C-score: 0.704983 (represented as a red bar for a standard deviation for C-scores).\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-4784472/v2/bcc8a4c68cc9480cf220ce7e.png"},{"id":71691237,"identity":"dd90b91e-dbef-4c3f-8fef-64953625ad85","added_by":"auto","created_at":"2024-12-17 18:16:48","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":184434,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 13: \u003c/strong\u003eThe 3D structure of the KRAS (Kirsten Rat Sarcoma Virus)\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-4784472/v2/60ba78b23fa23c3319fd41f0.png"},{"id":71691236,"identity":"d8620421-688c-4bde-8278-0a0a1c05fedd","added_by":"auto","created_at":"2024-12-17 18:16:48","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":265328,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 14: \u003c/strong\u003eThe solvent accessibility score of the KRAS (Kirsten Rat Sarcoma Virus)\u003c/p\u003e\n\u003cp\u003eThe figure presents the solvent accessibility score of the KRAS protein sequence. The sequence is displayed along the top, ranging from residues 1 to 180. Below the sequence, the predicted solvent accessibility scores are provided, where values range from 0 to 9. A score of 0 indicates a buried residue, while 9 signifies a highly exposed residue. This visual representation helps in understanding the exposure level of each residue in the KRAS protein, which is crucial for insights into its structure and interaction with other molecules.\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-4784472/v2/c903232890dc232527d15fca.png"},{"id":71692251,"identity":"56197ceb-0cb6-4e77-be36-e7f9f0bfc674","added_by":"auto","created_at":"2024-12-17 18:24:48","extension":"png","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":40478,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 15: \u003c/strong\u003eNormal Probability Plot. This figure displays a Normal Probability Plot of the predicted values against their sample percentiles. The predicted values follow a normal distribution.\u003c/p\u003e\n\u003cp\u003eThe horizontal axis represents the sample percentiles, while the vertical axis represents the predicted values. \u003cstrong\u003eData Points (Series 1):\u003c/strong\u003e The blue diamonds represent the observed predicted values plotted against their corresponding sample percentiles. \u003cstrong\u003eTrend Line (Linear Series1):\u003c/strong\u003e The black line represents the best-fit line for the data points. The equation of the line is y=0.0236xy = 0.0236xy=0.0236x and the coefficient of determination (R²) is 0.6028, indicating that approximately 60.28% of the variance in the predicted values can be explained by the sample percentiles.\u003c/p\u003e","description":"","filename":"15.png","url":"https://assets-eu.researchsquare.com/files/rs-4784472/v2/7c755e1266868e2469549e10.png"},{"id":71691244,"identity":"a9c0a261-7d89-4d32-adb1-c8e4a4702cdb","added_by":"auto","created_at":"2024-12-17 18:16:49","extension":"png","order_by":17,"title":"Figure 17","display":"","copyAsset":false,"role":"figure","size":93086,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 16: \u003c/strong\u003eThe thermodynamic states of the KRAS. The images are varied based on the values of the C score. The C Score values. (a) -0.02, (b) -1.18, (c) -1.37, (d) -0.92, and (e) -1.45.\u003c/p\u003e\n\u003cp\u003eHere, upwardly predicted five different models of the KRAS are drawn based on the C score value of the KRAS. Where (a) has a C score value of (-0.02) and is relatively more stable than model (e), which has a C score value of (-1.45). As for the model (b), (c), and (d), they have different C score values, respectively. The C-score value of model(b) is (-1.18), model (c) is (-1.37), and model (d) is (-.92). These models are very important to analyze the mutational status of the KRAS. Among the primary amino acid sequences, the G12S, G13C, Q61R, Q61K, and Q61X are very important for their mutational states. The different mutant features of the KRAS are shown in the image (p), and each of them is structurally different from the other, so as their specific actions.\u003c/p\u003e","description":"","filename":"16.png","url":"https://assets-eu.researchsquare.com/files/rs-4784472/v2/6db6ae73330a68b64f3f67c3.png"},{"id":71692253,"identity":"c5b25a39-85da-457f-b212-f3cad37025f7","added_by":"auto","created_at":"2024-12-17 18:24:49","extension":"png","order_by":18,"title":"Figure 18","display":"","copyAsset":false,"role":"figure","size":248870,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 17\u003c/strong\u003e: KRAS Mutation Overview; its related factors, analysis, technology, and signalling pathways.\u003c/p\u003e\n\u003cp\u003eThe diagram illustrates the elements associated with KRAS missense mutation, highlighting the related factors, analysis methodologies, technological tools, and signalling pathways. The central focus is on the KRAS gene, surrounded by several key components. The signalling pathways connected to KRAS include RAL-Nf-kB (highlighted in purple), RAF-MEK-ERK (highlighted in blue), and PI3K-AKT-mTOR (highlighted in black). Genetic and molecular factors associated with KRAS mutation are represented by CDKN2A (highlighted in red), SMAD (highlighted in red), NRAS (highlighted in yellow), HRAS (highlighted in black), P53 (highlighted in black), APC (highlighted in green), DCC (highlighted in yellow), and VMD (highlighted in red). Additionally, the diagram encompasses various analysis methodologies and technological tools such as Cluster Density (highlighted in orange), C-SCORE (highlighted in black), NAMD (highlighted in pink), and I-TESSER (highlighted in yellow). This comprehensive overview emphasizes the intricate network and interactions of KRAS with other genetic and molecular factors, and the relevant signaling pathways and technologies utilized for analysis.\u003c/p\u003e","description":"","filename":"17.png","url":"https://assets-eu.researchsquare.com/files/rs-4784472/v2/8e11cba812df1d3404065f85.png"},{"id":71692468,"identity":"86e539d0-81c7-4bac-b33a-abbf929ae30b","added_by":"auto","created_at":"2024-12-17 18:32:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4602440,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4784472/v2/70c12dcc-4db8-45c1-9d74-9c63d7c1f595.pdf"},{"id":71691239,"identity":"0130907d-d31f-43df-8fc4-3e3e4ef79999","added_by":"auto","created_at":"2024-12-17 18:16:48","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":67465,"visible":true,"origin":"","legend":"","description":"","filename":"Table01KRAS.docx","url":"https://assets-eu.researchsquare.com/files/rs-4784472/v2/25d1a6debdbeb955c568b9ba.docx"},{"id":71691222,"identity":"6dbcffa8-afe6-4740-a816-510481220b31","added_by":"auto","created_at":"2024-12-17 18:16:47","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14273,"visible":true,"origin":"","legend":"","description":"","filename":"Table02KRAS.docx","url":"https://assets-eu.researchsquare.com/files/rs-4784472/v2/af5eed3a512d2efe2044f0b9.docx"},{"id":71691230,"identity":"f73f74e8-45fa-4b37-9973-934d49a453b7","added_by":"auto","created_at":"2024-12-17 18:16:47","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":99113,"visible":true,"origin":"","legend":"","description":"","filename":"Table03KRAS.docx","url":"https://assets-eu.researchsquare.com/files/rs-4784472/v2/b6a9d53e7c324b1a663fc9df.docx"},{"id":71691240,"identity":"6ebeb34d-d4ec-4850-980f-c9c3f7acd6d7","added_by":"auto","created_at":"2024-12-17 18:16:49","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":108523,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-4784472/v2/d675269219fa79b19ad24b76.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"The Application of i-Tasser and related studies to predict the full-length structure, thermodynamic states and functional analysis of the Kirsten Rat Sarcoma Virus (KRAS) protein; An observation of KRAS structural and functional integrity.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eKRAS, the Kirsten Rat Sarcoma Virus, is a part of the Rat family Sarcoma Virus (RAS). It\u0026rsquo;s a signal transducer protein. That means it can regulate the various signaling pathways responsible for cellular survival, invasion, growth, and the functional regulation of various channels. KRAS is also a GTPase (Guanosine Triphosphate) activated enzyme-based RAS family oncoprotein. The structure of the KRAS is 85% homologous for the development of different types of Cancer in comparison to other isoforms of the Ras family (i.e.: Harvey Rat Sarcoma Virus, Kirsten Rat Sarcoma Virus and Neuroblastoma Rat Sarcoma Virus). That\u0026rsquo;s why it\u0026rsquo;s been regarded as the most significant among all of the RAS family onco-proteins. The bondage of the KRAS at the Receptor Tyrosine Kinase (RTK) or, more specifically, at the Effector Growth Factor Receptor (EGFR) leads to a higher intracellular concentration of the GTP than the GDP (Guanosine di-phosphate), which eventually activates the membrane-bound isopropyl based KRAS protein. Later, this activation of the KRAS stimulates the different cellular pathways such as [RAS- AKT (Adenosine Tyrosine Kinase)-mTOR] responsible for cellular proliferation, which means the growth of the cells, [RAL-Nf-kB] activates the innate immunity, necessary for the regulation of the cellular defense, and [RAF-MEK- ERK] maintains the life cycle of the cells. However, due to various genetic disorders, errors in the synthesis of the KRAS at the site of the ribosome, or the impact of external factors such as the Potential of Hydrogen (pH), temperature, and others, KRAS can go through a series of mutational challenges leading to the development of missense or point mutations. As a result, the Aspartic acid replaces Glycine, making the KRAS hyperactivated. So, whether a receptor bounds at the EGFR or not, KRAS will always remain hyperactivated, eventually impacting the transcription and the transmission to synthesize the myeloma cells or cells with infinite division and growth.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 01: \u0026nbsp;\u003c/strong\u003eThe occurrence of the Missense mutation (Glycine by Aspartic acid).\u003c/p\u003e\n\u003cp\u003eThis figure illustrates the mechanism leading to a missense mutation or point mutation in the KRAS gene. The primary sequence of the KRAS protein is shown, with a specific focus on the codon sequence where a mutation can occur. The transition from glycine to aspartic acid, highlighted in the sequence, is a common mutation site. External factors such as pressure, temperature, and dysregulation can induce this mutation. The process is depicted as a sequence of events starting from the primary KRAS sequence, influenced by external factors, leading to a missense mutation or point mutation. This mutation results in the substitution of glycine with aspartic acid, significantly impacting the protein\u0026apos;s function and contributing to oncogenic transformation.\u003c/p\u003e\n\u003cp\u003eThese mutations can lead to the development of various types of Cancer and metabolic disorders. So, to learn about various types of KRAS structures, we must understand their thermodynamic properties first. In the KRAS, the P loop or the Switch-I is the predominant site of mutations. Alongside 7 different mutational states, the application of engineered mutations seems highly frequent (40%). According to some recent analysis, almost 53.5% contain engineered mutations. WT (Wild type), C118S, G12A, G12D, G12V, and Q61H are common. We have performed the study in two completely different ways. At first, we conducted the study, where the first part of the analysis focused on the alterations of the KRAS structure and the development of various disorders. This covers both aspects of the physiological and the functional point of analysis. In the second part of the study, we have drawn the KRAS\u0026apos;s Primary (collected from the PDB), Secondary, 3D, and Thermodynamic states using the i-Tesser. Eventually provides us with a complete orientation of the KRAS structure and its functional characteristics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.2. Alterations of the KRAS structure and the functions\u0026nbsp;\u003c/strong\u003eUsually, the mutational transformation results from replacing the Glycine with Aspartic acid, leading to missense mutations or point mutations. [\u003cstrong\u003e\u003cu\u003eNote:\u003c/u\u003e\u003c/strong\u003e It\u0026rsquo;s a type of mutation in which a single amino acid from the residue gets replaced by another amino acid, resulting in various undesired side effects.]. As a result, hyperactivated KRAS is developed, which can function independently to continuously synthesize cellular division and survival. Additionally, the pH of the location gets lower, and it damages various cellular organelles and generates Reactive Oxygen Species (ROS), which is responsible for the occurrence and the resurgence of Cancers and various related disorders.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 02:\u003c/strong\u003e This schematic diagram illustrates the impact of the Rat Sarcoma (RAS) gene and its variants on different cell types and the associated conditions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.3. The Role of KRAS in the Development of Cancer\u003c/strong\u003e As discussed in the previous section, the KRAS mutational characteristics are responsible for tumor cell growth and the development of PDAC (Pancreatic Adenocarcinoma) and CRC (Colorectal Cancer). Metastases of these two types of solid tumors is the reason behind the mortality of thousands worldwide.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/58854_b38fc7f3db2c487f/58854_custom_files/img1722355239.png\" width=\"400\" height=\"249\"\u003e\u003c/p\u003e\n\u003cp\u003eIn this formula, S represents the standard deviation of the population, n is the size of the sample, xxx denotes each value from the given population, and x\u0026oline;overline{x}x is the mean of the population. In the context of KRAS mutation research, calculating the standard deviation is essential for understanding the variability and dispersion of data points in experimental results, such as gene expression levels or responses to treatments. This statistical tool aids in assessing the reliability and significance of the observed data, thereby contributing to the interpretation of experimental outcomes and the development of targeted therapies.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/58854_b38fc7f3db2c487f/58854_custom_files/img172235523911.png\" width=\"539\" height=\"268\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eThe table gives us an idea about Lung, Pancreatic, Blood, and Colon cancers and their rate of survival in recent times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 03:\u003c/strong\u003e Bar-chart to highlight the relationship between Cancer types and the Rate of Survival.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe bar diagram on the figure visually represents the given information from the provided table with a marginal value of standard deviation. Based upon the upwardly mentioned Table and the graphical representation of the data, the relationship between the different type of cancer and the survival rate in 2018 is resembled and points that even with the application of the various advanced treatments and therapies both Lung and Pancreatic cancer still has the highest morbidity, 95.1%, and 92%, respectively. Application of the standard deviation equation has helped us to figure out the Standard Deviation. KRAS plays a significant role in the occurrence of PDAC, having one of the highest morbidities, making it more interesting due to its sensitive location at the intersection of various blood vessels and with a success rate of 15% only the traditional therapies such as Radiotherapy and Chemotherapy are unable to function appropriately due to the rapid upregulation of alternative compensatory pathways as well as desmoplastic reaction ultimately making it more challenging.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo check the mutational and transcriptional landscape of the PDAC, the GTPase KRAS alongside tumor suppressor genes such as p53, CDKN2A (Cyclic-dependent kinase inhibitor type-2), and SMAD4(Mothers Against Decapentologenic-4) exhibited mutations. The mutated KRAS, amplification of their mutant alleles, overexpression of AKT-2, and the activity of the upstream regulator (PI3K) are often elevated in PDAC, increasing the survival of the tumor cells, which can be furtherly defined as a part of the epigenetic dysregulation resulted due to the modifications of the human DNA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 04:\u0026nbsp;\u003c/strong\u003eThe developmental flowchart of Pancreatic Adenocarcinoma (PDAC)\u003c/p\u003e\n\u003cp\u003eThe upward figure resembles the development of Pancreatic Adenocarcinoma (PDAC). As soon as the mutation at the KRAS results, it leads to the initiation of the Pancreatic intra-neoplasm stage 1, where the cells on the pancreas go through a series of altered modifications regarded as the initiation of tumorigenesis. The developed hyperplastic lesions are known as pancreatic intraepithelial neoplasia and the intraductal mucinous neoplasms (IPMN) and their prosperity to develop into cancer, which looks like finger (Papillae). Then it comes to stage 2 (mucinous cystic neoplasm) and stage 3 (intraductal papillary mucinous neoplasm) due to the inhibition of cyclic-dependent kinase 2A\u0026mdash;eventually, the development of the PDAC results from inhibiting the tumor suppressor gene p53 and SMAD-4. Similarly, the KRAS has also been responsible for the development of Colorectal Cancer. It is considered the most life-threatening among solid tumors and the third most diagnosed cancer in United States of America (USA). According to the latest prediction been made by the American Cancer Society, 153,020 will be diagnosed with cancers, and 52,550 will die in 2023.Similar to Pancreatic Cancer, the initiation of the Colorectal Cancer is also started in the normal epithelial cells. The inhibition of the Adenoma Polypmatous Coli (APC) develops an abnormal growth of the cells called; Adenomatous Polyp, impacted by the missense mutation because of the KRAS. The APC initiates most of both sporadic and hereditary colorectal cancers. The molecular outcome of the APC function loss lies mainly in its inability to downregulate the oncogenic canonical Wnt signaling pathway. The abnormal cascade activation leads to the overexpression of Wnt target genes involved in proliferation. Resulting in the development of lower-grade dysplasia or disordered cellular structures, develops Higher-grade Dysplasia at the inhibition of the DCC gene [\u003cstrong\u003eNote:\u003c/strong\u003e both cellular changes can easily be observed in the figure entitled \u0026ldquo;The physiological development of Colorectal cancer\u0026rdquo;], a putative tumor suppressor gene. The somatic mutations of the DCC gene and the netrin-1 during the organization of the spinal cord led to the position of DCC inactivation, significantly impacting tumorigenesis. To eventually inhibit the p53 gene, resulting in Colorectal Cancer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 05:\u0026nbsp;\u003c/strong\u003eThe developmental flowchart of Colorectal Cancer (CRC)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cstrong\u003eFigure 06:\u0026nbsp;\u003c/strong\u003e\u003c/strong\u003eThe KRAS regulated various cellular pathways.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.4. The Role of KRAS in the Development of Rasopathies\u0026nbsp;\u003c/strong\u003eThe hyperactivation of the KRAS can also lead to the development of Rasopathies. It\u0026rsquo;s a series of rare disorders where the alterations in the \u0026ldquo;Ras (Rat Sarcoma Virus)/MAPK (Mitogen Associated Phosphate Kinase)\u0026rdquo; signaling pathways occur. A table is given below to mention various KRAS dependent Rasopathies, their symptoms, and causes.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cstrong\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;'\u003eTable 02: KRAS-dependent Rasopathies, Symptoms, and their causes\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 155.8pt;border: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cstrong\u003e\u003cu\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;'\u003eRasopathies\u003c/span\u003e\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155.85pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cstrong\u003e\u003cu\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;'\u003eSymptoms\u003c/span\u003e\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155.85pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cstrong\u003e\u003cu\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;'\u003eCause\u003c/span\u003e\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 155.8pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;'\u003eNeurofibromatosis type 1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155.85pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;'\u003eNeurofibromas, problems with the eyes and the bones\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155.85pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;'\u003eNF-1 gene mutation\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 155.8pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;'\u003eNoonan Syndrome\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155.85pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;'\u003eShort stature, heart defects, unusual facial features.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155.85pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;'\u003eAltered KRAS, RAF-1 gene\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 155.8pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;'\u003eCardio-facio-cutaneous Syndrome\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155.85pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;'\u003eSmall chin and short nose\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 155.85pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:200%;'\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;'\u003eAltered KRAS, BRAF, MEK1/MEK2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.5. The Role of the KRAS in the Development of Diabetes Mellitus Type-2\u0026nbsp;\u003c/strong\u003eIn addition to Cancer and Rasopathies, the KRAS can also play a significant role in the development of Diabetes Mellitus Type-2. The bondage between the advanced glycolytic endpoints at the receptor activates the Phosphate Kinase C(PKC), eventually impacting the secretions of the NADPH oxidase. This increases the intercellular concentration of the Reactive Oxygen Species (ROS) and damages the DNA. All these results are due to the mutations in the KRAS and affect the secretion of the Cytochrome P450 and the Glycolysis process by inhibiting the Hexosamine kinase and Pentose Phosphate pathways. These alterations of the cascade develop insulin-resistant diabetes mellitus type-2, functioning in an unusual positive feedback mechanism rather than the natural positive feedback mechanism. A drop in the glucagon level in the blood leads to the rise of insulin concentrations in the peripheral tissues. This is an example of the negative feedback mechanism. However, in the positive feedback process, they work in an agonistic pattern by increasing each other\u0026rsquo;s volume, which seems to be synchronizing. That\u0026rsquo;s how the insulin-resistant diabetes develops.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 07:\u0026nbsp;\u003c/strong\u003eThe developmental process of the diabetes mellitus type-2, due to the mutation of the KRAS.\u003c/p\u003e\n\u003cp\u003eThe figure illustrates the pathway involved in the development of type-2 diabetes mellitus as a consequence of KRAS mutation. The process begins with Advanced Glycation End-products (AGE) interacting with the Receptor for Advanced Glycation End-products (RAGE), leading to the activation of Protein Kinase C (PKC). This activation stimulates NADPH oxidase, resulting in the production of Reactive Oxygen Species (ROS). The ROS, in turn, influences various metabolic processes, including the activity of cytochrome P450 and the regulation of glucose levels. ROS also impacts the pentose phosphate pathway and hexosamine kinase activity within the inner membrane, both of which are linked to glycolysis. The interconnected pathways highlight the complex biochemical cascade triggered by KRAS mutation, contributing to the pathogenesis of type-2 diabetes mellitus.\u003c/p\u003e"},{"header":"Methodology","content":"\u003cp\u003e\u003cstrong\u003eFigure 08:\u0026nbsp;\u003c/strong\u003eThe Primary Amino acid Sequence of the KRAS (Kirsten Rat Sarcoma Virus)\u003c/p\u003e\n\u003cp\u003eThe collected primary sequence of the Kirsten Rat Sarcoma Virus (KRAS) from the Protein Data Bank (PDB) [\u003cstrong\u003e\u003cu\u003eNote:\u003c/u\u003e \u003cu\u003ePDB ID:\u003c/u\u003e\u003c/strong\u003e 7X8N] is being furtherly analyzed by the application of the Bioinformatic tool \u0026ldquo;i-Tesser; Iterative threading Assembly Refinement.\u0026rdquo; \u0026nbsp;According to the critical assessment of techniques for protein structure prediction (CASP), the i-Tesser was awarded the first position for the CASP-7 and CASP-8. That\u0026rsquo;s the reason behind the choice of the i-Tesser. It focuses on the principles of the Monte Carlo Simulation technique for 3D visualization. It utilizes a combination between the \u0026ldquo;Nanoscale Molecular Dynamics (NAMD)\u0026rdquo; and \u0026ldquo;Visualization Molecular Dynamics (VMD).\u0026rdquo; The primary sequences of the protein get through the \u0026ldquo;Local meta-threading server (LOMETS).\u0026rdquo; to develop a template for the fragments. Later, the templates are assembled based on their increasing decoy-based optimization potential value. \u0026nbsp;The structure is usually clustered with the molecules with the lowest energy level developed by the Cluster centroid with the \u0026ldquo;SPICKER\u0026rdquo; software application. The same set of molecules is then reassembled, and from there, the molecules with the lowest energy structure show the optimization values of the H-bond to generate a finalized model. During this phase of 3Dstructural visualization, both the \u0026ldquo;Nanoscale Molecular Dynamics (NAMD)\u0026rdquo; and the \u0026ldquo;Visualization Molecular Dynamics (VMD)\u0026rdquo; are applied. This helps to simulate larger-size biomolecules ranging from 10,000 to 10,000,000 atoms, and the various rendering styles and methods differentiate between regions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 09:\u0026nbsp;\u003c/strong\u003eThe ligand-bound structure of the KRAS (Kirsten Rat Sarcoma Virus).\u003c/p\u003e\n\u003cp\u003eThe figure illustrates the three-dimensional structure of the KRAS protein in its ligand-bound state. The protein backbone is depicted in green, highlighting the intricate folding and secondary structural elements of the molecule. The bound ligand, visible within the structure, underscores key interaction sites critical for KRAS function and its role in signalling pathways. This structural representation provides valuable insights into the molecular interactions and conformational changes associated with ligand binding, aiding in the understanding of KRAS-related mechanisms and potential therapeutic targeting.\u003c/p\u003e\n\u003cp\u003eIn this way, the top 5 structural models and the ligand-bounded structures are estimated based on their C score values using the algorithmic ResQ analysis, indicating a model of global topology. Moreover, the i-Tesser helps to predict a comparatively stable form of the secondary structures. This has been further supported by the higher density of the amino acid molecules at the 2\u003csup\u003end\u003c/sup\u003e quadrant of the Ramachandran plot.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 10:\u0026nbsp;\u003c/strong\u003eThe Ramachandran Plot of the KRAS (Kirsten Rat Sarcoma Virus).\u003c/p\u003e\n\u003cp\u003eThe figure displays the Ramachandran plot for the KRAS protein structure, illustrating the distribution of phi (\u0026Phi;) and psi (\u0026Psi;) dihedral angles in the polypeptide chain. Each point on the plot represents a residue in the protein structure. The plot is divided into regions indicating the preferred conformations of the polypeptide backbone. The yellow areas denote allowed regions, while the red areas signify the most favoured regions. A notable feature in this plot is the left-handed beta-sheet structure, highlighted with a yellow arrow pointing towards the cluster of data points in the upper left quadrant, corresponding to the left-handed beta-sheet conformation. This plot provides insights into the conformational constraints and flexibility of the KRAS protein, crucial for understanding its functional dynamics.\u003c/p\u003e\n\u003cp\u003eThe B factor structure value of the KRAS and the solvent accessibility score are more detail-oriented values to explain the thermodynamic properties of the structural stability and the functional ability of the KRAS protein, which has been analyzed and drawn for the sake of the multiple angle analysis. In addition, we have utilized \u0026ldquo;\u003cstrong\u003eMicrosoft Excel-2022\u003c/strong\u003e\u0026rdquo; to analyze the statistical relationship between the \u0026ldquo;\u003cstrong\u003eC-Score and Cluster Density\u003c/strong\u003e.\u0026rdquo; and \u0026ldquo;the extent of comparative mortality between various forms of cancers.\u0026rdquo;\u0026nbsp;\u003c/p\u003e"},{"header":"Result","content":"\u003cp\u003eThe predicted secondary structure of the KRAS and their various structural forms are drawn based on their confidence score values, ranging from (\u003cstrong\u003e0-9\u003c/strong\u003e). The higher the means, the more confident the prediction of the structure.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 10:\u003c/strong\u003e KRAS confidence score values\u003c/p\u003e\n\u003cp\u003eSimilarly, the B factor structure values are estimated, referring to the thermal stability score having a range of (-2,5). The lower the value, the more stable the structures. So, the more negative the value more stable it is. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 11:\u0026nbsp;\u003c/strong\u003eThe B factor value of the KRAS (Kirsten Rat Sarcoma Virus)\u003c/p\u003e\n\u003cp\u003eAdditionally, the solvent accessibility score is estimated to depict the reactive aspect of the sequence. These values range between 0(buried residue) and 9(highly exposed residue). To analyze the relationship extent between 5 different models of the KRAS, both the C score and the Cluster density values are analyzed. Where the C-Score value lingers around (-0.02) to (-1.45), and the cluster density varies from (0.0683) to (0.2835), with a Standard Deviation (SD) value of (0.704983).\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/58854_b38fc7f3db2c487f/58854_custom_files/img1722355541.png\" width=\"765\" height=\"373\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003ctable style=\"border: none;width:478.85pt;border-collapse:collapse;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 150.15pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:red;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:red;'\u003eANOVA: Single Factor\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e(Confidence Score)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 39.5pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.35pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 21.65pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 88.9pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.85in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 39.5pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.35pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 21.65pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 88.9pt;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eSUMMARY\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.85in;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 39.5pt;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.35pt;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 21.65pt;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 88.9pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cem\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eGroups\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.85in;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cem\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eCount\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 39.5pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cem\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eSum\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.35pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cem\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eAverage\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.95in;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cem\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eVariance\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 21.65pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 88.9pt;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.85in;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 39.5pt;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e14\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.35pt;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e3.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e1.666667\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 21.65pt;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 88.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e-0.02\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.85in;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 39.5pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e-4.92\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.35pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e-1.23\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.95in;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e0.055533\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 21.65pt;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 88.9pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.85in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 39.5pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.35pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 21.65pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 88.9pt;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.85in;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 39.5pt;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.35pt;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 21.65pt;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 88.9pt;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eANOVA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.85in;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 39.5pt;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.35pt;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 21.65pt;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 88.9pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cem\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eSource of Variation\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.85in;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cem\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eSS\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 39.5pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cem\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003edf\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.35pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cem\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eMS\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.95in;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cem\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eF\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.95in;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:red;'\u003eP-value\u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.95in;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cem\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eF crit\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.65pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 88.9pt;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eBetween Groups\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.85in;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e44.7458\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 39.5pt;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.35pt;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e44.7458\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e51.96354\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e0.000361\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e5.987378\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.65pt;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 88.9pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eWithin Groups\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.85in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e5.1666\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 39.5pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e6\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.35pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e0.8611\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 21.65pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 88.9pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.85in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 39.5pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.35pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 21.65pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 88.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eTotal\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.85in;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e49.9124\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 39.5pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.35pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.95in;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.95in;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.95in;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 21.65pt;padding: 0in 5.4pt;height: 16.35pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 88.9pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.85in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 39.5pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.35pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0.95in;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 21.65pt;padding: 0in 5.4pt;height: 15.85pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:200%;'\u003e\u003cspan style='line-height:200%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border: none;width:11.5pt;border-collapse:collapse;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 11.5pt;padding: 0in 5.4pt;height: 13.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:red;'\u003eANOVA: Single Factor\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e(Cluster Density)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003ctable style=\"border: none;width:467.55pt;border-collapse:collapse;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81.5pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 49pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81.5pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 49pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 143.95pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 49pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81.5pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 49pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81.5pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eSUMMARY\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 49pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81.5pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003eGroups\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003eCount\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003eSum\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003eAverage\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003eVariance\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81.5pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e14\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e3.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e1.666667\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81.5pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e0.2835\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e0.3468\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e0.0867\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e0.000439\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81.5pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 49pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81.5pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 49pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81.5pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eANOVA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 49pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81.5pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003eSource of Variation\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003eSS\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003edf\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003eMS\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003eF\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:red;'\u003eP-value\u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003eF crit\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81.5pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eBetween Groups\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e23.30123\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e23.30123\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e27.95412\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e0.001852\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e5.987378\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81.5pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eWithin Groups\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e5.001317\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e6\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e0.833553\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81.5pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 49pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81.5pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eTotal\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e28.30255\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.75pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 81.5pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 49pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 62.45pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 12.4pt;padding: 0in 5.4pt;height: 12.4pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eBoth ANOVA analyses (for C-scores and Cluster Density) indicate statistically significant differences between the groups. The F-values in both cases are much larger than the F critical values, and the P-values are significantly lower than 0.05. This suggests that the structural models of KRAS have significantly different C-scores and Cluster Density values.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 12:\u0026nbsp;\u003c/strong\u003eKRAS Structural Stability.\u003c/p\u003e\n\u003cp\u003eThis bar graph illustrates the relationship between five different structural models of the KRAS protein, C-scores, and Cluster Densities. The C-scores are represented by red bars, while the Cluster Densities are represented by black bars. The graph also includes trend lines for both C-scores (dotted blue line) and Cluster Densities (dotted orange line). \u003cstrong\u003eModel 01\u003c/strong\u003e:C-score: -0.02, Cluster Density: 0.2835, \u003cstrong\u003eModel 02\u003c/strong\u003e: C-score: -1.18, Cluster Density: 0.0895, \u003cstrong\u003eModel 03\u003c/strong\u003e: C-score: -1.37, Cluster Density: 0.0739, \u003cstrong\u003eModel 04\u003c/strong\u003e: C-score: -0.92, Cluster Density: 0.1151, \u003cstrong\u003eModel 05\u003c/strong\u003e: C-score: -1.45, Cluster Density: 0.0683, \u003cstrong\u003eStandard Deviation (SD)\u003c/strong\u003e: C-score: 0.704983 (represented as a red bar for a standard deviation for C-scores).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe graph shows that Model 01 has the highest Cluster Density and the least negative C-score, indicating potentially better structural stability compared to the other models. Model 05 has the lowest C-score and Cluster Density, suggesting it might be the least stable. The trend lines provide a visual representation of the overall trends in C-scores and Cluster Densities across the models. This graph provides insight into the structural stability of different KRAS protein models, with implications for their reliability in research and potential therapeutic applications. Higher Cluster Density and less negative C-scores are indicative of more reliable structural models.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe KRAS is a 189 amino acid-long protein substance. The molecular weight of the KRAS is 21KDa, which has no structural sub-unit. It has 5 alpha helices and 6 beta-strand; it\u0026rsquo;s one of the three isoforms of the RAS family protein. Been divided into two major regions\u0026mdash;G domain (1-166) amino acid chain and Hypervariable region (HVR). lasts from (167-189) amino acids. The KRAS\u0026apos;s generalized primary amino acid sequence has a specialized structural motif called; the P loop. This is also popularized as the \u0026ldquo;Walker A motif.\u0026rdquo;, the structural motif is highlighted by the green color. Where we can observe the existence of the primary amino acid sequence, a pattern of \u0026ldquo;\u003cstrong\u003eGAAAAGKS\u003c/strong\u003e,\u0026rdquo; Where the A stands for any amino acid molecule.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 13:\u0026nbsp;\u003c/strong\u003eThe 3D structure of the KRAS (Kirsten Rat Sarcoma Virus)\u003c/p\u003e\n\u003cp\u003eUusally, the P-loop is a structural pocket where the proteins can easily bind. The G domain can be furtherly subdivided into 3 different parts; P-loop (10-17), Switch-I (30-40), and Switch-II (59-76). Similar to the other related mutational hotspots, such as; Switch-I and Switch-II, are highlighted with red and purple colors respectively. As for the C terminal-based Hypervariable region, it\u0026rsquo;s been showcased with a navy blue color. The KRAS has 2 isoforms [\u003cstrong\u003e\u003cu\u003eNote:\u003c/u\u003e\u003c/strong\u003e A chemically active compound generated from the same prtoein] KRAS4A and KRAS4B, differentiated at the following sites; 151,153,165,166 and the Hypervariable regions. For the necessity the HVR, lately can go through the following post-translational modifications; Farnesylation, Methylation, and Palmitoylation. Insertion of the primary amino acid sequence in the i-Tesser resulted in the prediction of the secondary protein structure. The higher the confidence score, the more reliable the predicted structure. A score of 9 meaning the secondary structure of the specific amino acid of the KRAS is more stable and realible. The Helix, Strand, and Coil structures are showcased with different colors. Which indicates that the predicted coiled from of the structure seems to be structurally more stable than the Helix and Strand conformations.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe Ramachandran plot supports the secondary structure of the protein, defining the torsion angle between the \u0026ldquo;psi\u0026rdquo; and \u0026ldquo;phi.\u0026rdquo; bond between the Carbon atoms. To make an accurately predicted protein structure, here, the PDBsum server is used to generate the Ramachandran plot. Where the greater density of the amino acids at the 2nd quadrant of the Ramachandran plot denotes the beta-stranded left-handed protein conformation.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eFinally, with the application of the NAMD and VMD principles, the i-Tesser will help us to draw the 3D structure of the KRAS. An ideal 3D structure of the KRAS is shown in the image. Where the pink color depicts the alpha-helices as a twisted structure. Whereas, the beta-strand is highlighted with the yellow color. The end of the C terminus is the White-colored one and \u0026nbsp;just under the C terminus, it\u0026rsquo;s the Hypervariable region. To check the thermal mobility of the KRAS protein, a frequency curve been drawn to provide the details about the structural stability of the amino acid residues. Normalized B factor value for a target protein is predicted by applying template-based assignment and profile-based prediction. The residues with higher than \u0026ldquo;0\u0026rdquo; B-factor values are comparatively less stable in experimental conditions. So, the negative values of the amino acid residues mean a comparatively more stable structure. The strand structures of the amino acid residues highlighted with green having a lower value, so they are structurally more stable. The helix and coil structures highlighted with purple and black colors are structurally either helix or coiled-shaped. To furtherly highlight the thermodynamic and structural characteristics of the predicted structure, we have performed the solvent accessibility score analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 14:\u0026nbsp;\u003c/strong\u003eThe solvent accessibility score of the KRAS (Kirsten Rat Sarcoma Virus)\u003c/p\u003e\n\u003cp\u003eThe figure presents the solvent accessibility score of the KRAS protein sequence. The sequence is displayed along the top, ranging from residues 1 to 180. Below the sequence, the predicted solvent accessibility scores are provided, where values range from 0 to 9. A score of 0 indicates a buried residue, while 9 signifies a highly exposed residue. This visual representation helps in understanding the exposure level of each residue in the KRAS protein, which is crucial for insights into its structure and interaction with other molecules.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 15:\u0026nbsp;\u003c/strong\u003eNormal Probability Plot. This figure displays a Normal Probability Plot of the predicted values against their sample percentiles. The predicted values follow a normal distribution.\u003c/p\u003e\n\u003cp\u003eThe horizontal axis represents the sample percentiles, while the vertical axis represents the predicted values. \u003cstrong\u003eData Points (Series 1):\u003c/strong\u003e The blue diamonds represent the observed predicted values plotted against their corresponding sample percentiles. \u003cstrong\u003eTrend Line (Linear Series1):\u003c/strong\u003e The black line represents the best-fit line for the data points. The equation of the line is y=0.0236xy = 0.0236xy=0.0236x and the coefficient of determination (R\u0026sup2;) is 0.6028, indicating that approximately 60.28% of the variance in the predicted values can be explained by the sample percentiles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003ctable style=\"border: none;width:6.5in;border-collapse:collapse;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 119.15pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003eRegression Statistics\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.8pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 54.45pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.35pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.7pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 71.5pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eMultiple R\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.65pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e0.354578\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.8pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 54.45pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.35pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.7pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 71.5pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eR Square\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.65pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e0.125726\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.8pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 54.45pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.35pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.7pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 71.5pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eAdjusted R Square\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.65pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e0.120786\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.8pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 54.45pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.35pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.7pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 71.5pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eStandard Error\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.65pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e1.405166\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.8pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 54.45pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.35pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.7pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 71.5pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eObservations\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.65pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e179\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.8pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 54.45pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.35pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.7pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 71.5pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 47.65pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 56.8pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 54.45pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.35pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.7pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 71.5pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eANOVA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.65pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 56.8pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 54.45pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.35pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.7pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 71.5pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.65pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003edf\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.8pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003eSS\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003eMS\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003eF\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.45pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003eSignificance F\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.35pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.7pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 71.5pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eRegression\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.65pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.8pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e50.25799\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e50.25799\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e25.45363\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.45pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e1.12E-06\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.35pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.7pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 71.5pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eResidual\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.65pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e177\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.8pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e349.485\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e1.974492\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 54.45pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.35pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.7pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 71.5pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eTotal\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.65pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e178\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e399.743\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.45pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.35pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.7pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 71.5pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 47.65pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 56.8pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 54.45pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.35pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.7pt;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 71.5pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.65pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003eCoefficients\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.8pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003eStandard Error\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003et Stat\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:red;'\u003eP-value\u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.45pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003eLower 95%\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003eUpper 95%\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51.35pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003eLower 95.0%\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51.7pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cem\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:black;'\u003eUpper 95.0%\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 71.5pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003eIntercept\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.65pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e1.849373\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.8pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e0.212702\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e8.694652\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:red;'\u003e2.31E-15\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.45pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e1.429614\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e2.269132\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51.35pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e1.429614\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51.7pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e2.269132\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 71.5pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47.65pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e-0.01025\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e0.002033\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e-5.04516\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-family: \"Times New Roman\",serif;color:red;'\u003e1.12E-06\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.45pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e-0.01427\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e-0.00624\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51.35pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e-0.01427\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51.7pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 14.65pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:right;line-height:normal;'\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e-0.00624\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 71.5pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 47.65pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 56.8pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 54.45pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 44.85pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.35pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 51.7pt;padding: 0in 5.4pt;height: 14.25pt;vertical-align: bottom;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eThe lower score means less exposure, and the higher score means relatively more exposed regions. The greater the exposure, the larger the surface volume ratio, making it more suitable to drug-protein conjugation. \u0026nbsp;As for the drug-protein conjugation, the overall structure of the KRAS is shown with the green color and the bounded molecules it\u0026rsquo;s been highlighted with different other colors. The conjugation always depicts the thermodynamic states of the KRAS\u0026rsquo;s 3D structural models.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 16:\u0026nbsp;\u003c/strong\u003eThe thermodynamic states of the KRAS. The images are varied based on the values of the C score. The C Score values. (a) -0.02, (b) -1.18, (c) -1.37, (d) -0.92, and (e) -1.45.\u003c/p\u003e\n\u003cp\u003eHere, upwardly predicted five different models of the KRAS are drawn based on the C score value of the KRAS. Where (a) has a C score value of (-0.02) and is relatively more stable than model (e), which has a C score value of (-1.45). As for the model (b), (c), and (d), they have different C score values, respectively. The C-score value of model(b) is (-1.18), model (c) is (-1.37), and model (d) is (-.92). These models are very important to analyze the mutational status of the KRAS. Among\u0026nbsp;the primary amino acid sequences, the G12S, G13C, Q61R, Q61K, and Q61X are very important for their mutational states. The different mutant features of the KRAS are shown in the image (p), and each of them is structurally different from the other, so as their specific actions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Limitations of this study and the related future research opportunities\u0026nbsp;\u003c/strong\u003eAlthough the authors have given different thermodynamic states of the KRAS or the mutant states of the KRAS, they are still required to provide the structural point of view of all 7 different mutant states of the KRAS protein. Also, the confidence score value among the amino acid residue is unequal, and this score usually varies. That indicates a level of uncertainty among the predicted structures. Additionally, the dynamics of the KRAS\u0026apos;s dimeric and oligomeric complexes are furtherly needed to be researched. \u0026nbsp;So, to resolve these challenges, state art models such as the Markov State Model (MSM) with a combination of the Cryo-EM (based on the principles of the NAMD and VMD) must be implemented.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, As a gene, KRAS plays a central role in the initiation, invasion, treatment, resistance, and reoccurrence of Colorectal and Pancreatic Cancers. A detailed idea about the KRAS and its functional and structural overview deciphers the opportunity to focus on this \u0026ldquo;Undruggable Oncoprotein\u0026rdquo;. So to investigate thoroughly about the KRAS, we have tried to focus on its conformational interest. This is necessary to analyze in the upcoming times to resolve the full-length structural and thermodynamic challenges associated with the KRAS.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interest:\u0026nbsp;\u003c/strong\u003eThe author declared that he has no known competing financial or personal relationships that could have appeared to influence the work reported in this research dissertation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e All the authors mutually agreeing for the publication this this study\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions:\u003c/strong\u003e the generation of the data to the interpretation and ultimately the development of the manuscript, each part of the complete manuscript been developed by DK under the guidance of MK (Mrinal Kranti Saha), Where the rest of the authors TC (Tanvir Chowdhury) have contributed especially to the screening and image visualization.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e The author would like to thank the University of Texas Rio Grande Valley, I-Tesser tool, and its developer Dr. Zhang et.al. from University of Michigan, Ann Arbor. We would furtherly like to share our gratitude towards our family members and well-wishers for their continuous support in developing this precious research manuscript and the author DK wants to share his special thanks to First Lutheran Church, Brookings, South Dakota-57006 for their hospitality.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAl Qahtani WS (2020) Utility of KRAS Gene and Clinicopathological Features in the Assessment of the Risk of Type 2 Diabetes in the Etiology of Colon Cancer. Global Medical Genetics\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuscial L (2020) Role of oncogenic KRAS in diagnosing, prognosis, and treating pancreatic cancer. Nature Reviews Gastroenterology and Hepatology\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCancer, Facts, Figures (2018) American Cancer Society\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eC.Phillips J (2020) Scalable molecular dynamics on CPU and GPU architectures with NAMD. J Clin Phys, 153\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD., S. (2017) LiteMol suite: Interactive web-based visualization of large-scale macromolecular structure data. Nature Methods\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHusic E, B (2018) Markov State Models: From an Art to a Science. Journal of American Chemical Society\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerrer I (2018) KRAS-Mutant non-small cell lung cancer: From biology to therapy. Lung Cancer\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeel F, N (2011) The RalGEF-Ral Effector Signaling Network: The Road Less Traveled for Anti-Ras Drug Discovery. Gene Cancer, 3(2)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrenknel D (2002) Understanding Molecular Simulation, vol 2. Elsevier\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarzilli I (2018) Design principles of the paradoxical feedback between pancreatic alpha and beta cells. Sci Rep, 8\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHillig Christian Roman (2022) Targeting RAS oncogenesis with SOS1 inhibitors. Advances in Cancer Research, vol 153. Elsevier\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang L (2021) KRAS mutation: From undruggable to druggable in cancer. Signal Transduction and Targeted Therapy\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK.Shimanshu D (2017) RAS Proteins and Their Regulators in Human Disease. Cell, p 170\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFriedman M (2022) J. Neurofibromatosis 1. Gene Reviews\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNoonan syndrome (2023) National Center for Advancing Translational Sciences\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNy\u0026iacute;ri K (2020) Structure-based inhibitor design of mutant RAS proteins\u0026mdash;A paradigm shift. Cancer Metastasis Rev, 39\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrth M (2019) Pancreatic ductal adenocarcinoma: Biological hallmarks, current status, and future perspectives of combined modality treatment approaches. Radiat Oncol, 14\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePanstar T (2020) The current understanding of KRAS protein structure and dynamics. Comput Struct Biotechnol, 18\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrakash P (2013) Lessons from computer simulations of Ras proteins in solution and in membrane. Biochimica et Biophysica Acta (BBA) - General Subjects, 1830\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eProtein Data Bank (2023)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRebecca L, Siegel MPH, PhD NSWMMHA, Andrea Cercek MD, Robert A Smith PhD, Ahmedin Jemal DVM, PhD. (2023). Colorectal cancer statistics, 2023. A Journal Cancer for the Clinicians\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoberts A (2006) The cardiofaciocutaneous syndrome. J Med Genet, 4\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePunekar R, S (2022) The current state of the art and future trends in RAS-targeted cancer therapies. Nat Reviews Clin Oncol, 19\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShibata H (2017) Mucinous Cystic Neoplasms Lined by Abundant Mucinous Epithelium Frequently Involve KRAS Mutations and Malignant Progression. Anticancer Research\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimon K (2016) Colorectal cancer development and advances in screening. Clin Invest Aging, 11\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStephanie PM (2018) RAS variant signaling. Biochem Soc Trans, 46(5)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eT.Ihle N (2012) Effect of KRAS Oncogene Substitutions on Protein Behavior: Implications for Signaling and Clinical Outcome. Journal of the National Cancer Institute\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang K-B (2022) Structural insight into the bulge-containing KRAS oncogene promoter G-quadruplex bound to berberine and coptisine. Nature Communications\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWiltgen M (2019) Algorithms for Structure Comparison and Analysis: Homology Modelling of Proteins. Encyclopedia of Bioinformatics and Computational Biology, 1\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang J, Yang J, Zhang Y (2015) I-TASSER server: New development for protein structure and function predictions. Nucleic Acids Research, 43: W174-W181 (2015). Nucleic Acid Research\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang M (2019) The structural basis for Ras activation of PI3Kα lipid kinase. Physical Chemistry Physical Physics\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng W (2021) Folding non-homologous proteins by coupling deep-learning contact maps with I-TASSER assembly simulations. Cell Reports Methods\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Rajshahi","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"KRAS, Kirsten Rat Sarcoma Virus, i-Tesser, Iterative Threading Assembly Refinement, C-Score, Confidence Score, Cluster Density, Holy Grail","lastPublishedDoi":"10.21203/rs.3.rs-4784472/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4784472/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eKRAS (Kirsten Rat Sarcoma Virus) is a Ras (Rat Sarcoma virus) family homologous. Due to its structural characteristics and biochemical properties, it’s been regarded as one of the most common drivers in various cellular signaling pathways. Due to its biochemical and pharmacological importance, KRAS been regarded as the “Holy Grail” of drug discovery. To understand the structure and thermodynamics of this protein, we tried to focus on the confirmational and functional portion of this specific protein. “i-Tesser” is applied to predict the structure of KRAS. A prediction for the secondary and full-length 3D structure been developed. Additional generation of the B factor value, Solvent accessibility, ligand bonded structure, as well as the 5 different thermodynamic state models of the KRAS and their functions are analyzed. The primary, metastable, ligand-bounded reactive states are visualized with the additional application of NAMD (Nanoscale Molecular Dynamics) and VMD (Visualization Molecular Dynamics).\u003cu\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/u\u003eThe drawn models showcase a C score value ranging between (-0.02 to -1.45) and Cluster density (0.0683 to 0.2835), along with a standard deviation value of (0.704983). Predicted secondary, 3D, and Thermodynamic models are drawn and supported by modeling their Solvent accessibility and B factor values. The 5 different models and the related structural and functional analysis provide additional information about the stability of KRAS structure and its function.\u003c/p\u003e","manuscriptTitle":"The Application of i-Tasser and related studies to predict the full-length structure, thermodynamic states and functional analysis of the Kirsten Rat Sarcoma Virus (KRAS) protein; An observation of KRAS structural and functional integrity.","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2024-12-17 18:16:42","doi":"10.21203/rs.3.rs-4784472/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2024-07-30 16:22:56","doi":"10.21203/rs.3.rs-4784472/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7329a737-14d1-47f8-86eb-4af96afe8a47","owner":[],"postedDate":"December 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":35355438,"name":"Structural Biology"}],"tags":[],"updatedAt":"2024-07-30T16:22:56+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-17 18:16:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-4784472","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4784472","identity":"rs-4784472","version":["v2"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}