In Silico Exploration of Nitrogenase in Ensifer fredii Unraveling Structural Homology Functional and Phylogenetic Relationships and Molecular Dynamics  

In Silico Exploration of Nitrogenase in Ensifer fredii Unraveling Structural Homology Functional and Phylogenetic Relationships and Molecular Dynamics | 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 In Silico Exploration of Nitrogenase in Ensifer fredii Unraveling Structural Homology Functional and Phylogenetic Relationships and Molecular Dynamics Mohamed Hnini, Karim Rabeh This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5209284/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract The nitrogenase Fe protein, central to nitrogen fixation, has been extensively studied, However, the X-ray structure of the Nase-Fe protein from Ensifer fredii is essential for understanding its function and stability, yet this structure is absent from the database. This study aimed to analyze the structural features of the NifH protein (EC 1.18.6.1, nitrogenase component II)) from Ensifer fredii using an in silico approach. Utilizing diverse biocomputational tools, we examined the primary, secondary, and tertiary structures of 22 NifH proteins, focusing on QSZ40499. Results indicate that QSZ40499 is a highly stable, hydrophilic, cytoplasmic protein with a molecular weight of 25.34 kDa. Its functional motif corresponds to the Fe4 family domain, and its secondary structure comprises 34.89% random coils and 34.47% alpha helices. 3D modeling via AlphaFold was validated using structural quality assessment servers and refined using the 3DRefiner server, supported by Z-scores and ERRAT Factor metrics. Molecular dynamics simulations revealed that QSZ40499 maintains structural stability and functional flexibility, essential for dynamic interactions during nitrogen fixation. This work provides valuable insights into the structural attributes of nitrogenase enzymes, advancing bioinformatics research and understanding of nitrogen fixation mechanisms. Nitrogenase Ensifer fredii Modeling Alphafold In silico MD-simulation Figures Figure 1 Figure 2 Article Highlights This study analyzes the Nitrogenase sequence gene (QSZ40499) from Ensifer fredii , emphasizing stability and functionality. 22 NifH proteins undergo a thorough examination of primary, secondary, and tertiary structures using diverse biocomputational tools. QSZ40499 demonstrates high stability, hydrophilicity, and cytoplasmic localization, with a molecular weight of 25.34 KD. Secondary structure analysis reveals a predominant random coil (34.89%) and alpha helix (34.47%) in the NifH protein. Alphafold is a reliable tool for NifH protein modeling, and the 3Drefiner server is suitable for subsequent refinement, contributing valuable insights to bioinformatics research. Molecular dynamics simulations show that the nitrogenase Fe protein maintains structural stability while possessing essential flexibility for its functional role in nitrogen fixation. 1. INTRODUCTION The primary source of fixed nitrogen (N) in the global biogeochemical cycle is biological conversion to ammonia (NH), facilitated by the microbial enzyme Nitrogenase [ 1 , 2 ]. Three types of nitrogenase enzymes have been identified: Molybdenum (Mo)-dependent, vanadium (V)-dependent, and iron (Fe)-dependent [ 3 – 5 ]. These enzymes are encoded by distinct gene clusters known as nif , vnf , and anf [ 6 – 8 ]. The extensively studied Mo-nitrogenase studied variant comprises two components: Fe proteins encoded by nifH genes and MoFe proteins encoded by nifD and nifK genes [ 9 ]. Protein structure modeling serves as a method to comprehend biological processes at the molecular level. While structural information on NifH proteins (EC 1.18.6.1, nitrogenase component II)) is known for some bacteria, such as Azotobacter , Clostridium , and Arthrobacter sp. [ 10 – 13 ], it is lacking for Ensifer sp. This can be explained by two factors: the lack of X-ray crystallographic research on the nitrogenase iron protein produced by Ensifer sp. and the absence of reliable three-dimensional (3D) modeling of the NifH protein. Understanding a protein's function necessitates knowledge of its 3D structure. Experimental determination through X-ray crystallography or NMR spectroscopy proves challenging, complicated, expensive, and time-consuming [ 14 , 15 ]. Addressing this, computational approaches, or alternative methods, particularly homology modeling, have gained widespread acceptance for predicting in silico 3D protein structures [ 16 ]. This study uses various biocomputational methods to characterize the enzyme Nitrogenase from Ensifer sp., an understudied subject. The investigation explores the structural, physicochemical, phylogenetic, and functional aspects of the nifH protein from Ensifer fredii . This specific strain was isolated from the nodules of Vachellia tortilis subsp. raddiana , a resilient leguminous tree thriving naturally in a challenging arid climate. In silico protein analysis is gaining popularity among scientists due to its significant contribution to obtaining baseline data in a timely and appropriate manner. This study will aid researchers in gaining a foundational understanding of this crucial enzyme, a member of the Rhizobiaceae family, and explores additional research directions, such as building a complex protein docking despite the complexity of symbiotic processes and nitrogen fixation mechanisms. This study is guided by the following research questions: What are the key structural and functional features of the nitrogenase Fe protein (NifH) in Ensifer fredii as determined through in silico methods? How does the structure of the NifH protein from Ensifer fredii compare to other homologous proteins across bacterial species in terms of phylogenetics and functionality? What insights can molecular dynamics simulations provide about the stability and flexibility of the NifH protein during nitrogen fixation? Based on these questions, the study hypothesizes that the nitrogenase Fe protein (NifH) in Ensifer fredii , modeled using advanced in silico approaches, demonstrates a unique combination of structural stability and functional flexibility essential for efficient nitrogen fixation. Specifically, the structural stability ensures the protein maintains its conformational integrity under physiological conditions, while the functional flexibility enables dynamic conformational changes necessary for its catalytic role in converting atmospheric nitrogen to ammonia. This balance between stability and flexibility is likely conserved across homologous proteins, reflecting an optimized functional approach for nitrogen fixation in diverse bacterial species. 2. MATERIAL AND METHODS 2.1. Sequence Retrieval, Alignment, and Phylogenetic Analysis The nitrogenase Fe protein, derived from the 'NifH' gene in Ensifer fredii , was isolated from the root nodule of Vachellia tortilis subsp. raddiana (QSZ40499). Using the NCBI database, the NifH amino acid partial sequence (accession no. QSZ40499.1) was obtained in FASTA format and employed as a BLAST query against a nonredundant protein database. For subsequent analysis, a subset of 22 sequences with an E-value below 4.30e-173 was selected. Multiple sequence alignments were performed using Clustal Omega ( https://www.ebi.ac.uk/jdispatcher/msa/clustalo ), encompassing deduced amino acid and cDNA sequences. The construction of a phylogenetic tree using MEGAX [ 17 ] through the maximum likelihood technique, supported by one thousand bootstrap replicates, resulted in two distinct trees representing amino acid sequences and cDNA from various Ensifer fredii nitrogenase strains. 2.2. Physicochemical Characterization The ExPaSy ProtParam tool ( https://web.expasy.org/protparam/ ) facilitated the determination of physicochemical characteristics for nitrogenase Fe protein sequences. These parameters included the grand average of hydropathogenicities (GRAVY), molecular weight (Mw), isoelectric point (pI), extinction coefficient (EC), instability index (II), and aliphatic index (AI). Prediction of the family domain for each protein was executed using ScanProsite ( http://prosite.expasy.org/scanprosite/ ), PFAM ( http://pfam.xfam.org ), and the MOTIF search ( https://www.genome.jp/tools/motif ) [ 18 ]. Subcellular structures were localized through Cello ( http://cello.life.nctu.edu.tw ) and PSLpred [ 19 ]. 2.3. Structure Prediction and Evaluation The secondary structure of amino acid sequences was predicted by SOPMA ( https://npsa-prabi.ib,cp.fr/cgi bin/npsa_automat.pl?page = npsa_sopma.html) and PSIPRED ( http://bioinf.cs.ucl.ac.uk/psipred/ ). As the 3D model template for our query sequence was unavailable in the Protein Data Bank (PDB), PHYRE2 [ 20 ], trRosetta ( https://yanglab.nankai.edu.cn/trRosetta/ ) [ 21 ], RaptorX ( http://raptorx.uchicago.edu/ ) [ 22 ], AlphaFold and AlphaFold II were utilized to construct homology 3D structures of five nitrogenase proteins, including our protein (Accession no. QSZ40499.1). Visualization of the model was conducted using Discovery Studio, version 21.1.0.[ 23 ]. Evaluation and verification of the predicted protein model of Ensifer fredii Nitrogenase was performed using the SAVES ( https://servicesn.mbi.ucla.edu/SAVES/ ) server. This involved various tools, such as the Ramachandran plot generated by the RAMPAGE server ( http://mordred.bioc.cam.ac.uk/rapper/rampage.php ) [ 24 , 25 ], Verify3D [ 26 ], and ERRAT for the examination of crystallographic structures. 2.4. Model Refinement To refine the predicted 3D protein model, three servers were utilized: 3Drefine protein structure refinement server ( http://sysbio.rnet.missouri.edu/3Drefine ) [ 27 ], DeepRefiner protein structure refinement server ( http://watson.cse.eng.auburn.edu/DeepRefiner ) [ 28 ], and GalaxyWEB web server ( http://galaxy.seoklab.org ) [ 29 ]. The refined nitrogenase protein models underwent evaluation and verification using the SAVES server ( http://services.mbi.ucla.edu/SAVES/ ), which included assessments through the Ramachandran plot, verify3D and ERRAT. 2.5. Molecular Dynamics Simulation To further assess the stability and behavior of the refined nitrogenase Fe protein model, a molecular dynamics (MD) simulation was performed using GROMACS, facilitated by the Visual Dynamics platform ( https://visualdynamics.fiocruz.br/ ) [ 30 ]. The simulation was executed in an APO state, employing the AMBER99SB-ILDN force field to ensure accurate modeling of protein dynamics. The system was solvated in a TIP3P water box, maintaining a 1.0 nm buffer from the protein to the box boundaries, and neutralized with counterions. The simulation environment was adjusted to mimic biologically relevant conditions: Temperature: Maintained at 300 K using the Nose-Hoover thermostat. Pressure: Maintained at 1 bar using the Parrinello-Rahman barostat. The simulation was conducted with a timestep of 2 femtoseconds (fs), following standard GROMACS protocols. Prior to the production run, energy minimization was performed to eliminate steric clashes, followed by equilibration in NVT (constant volume and temperature) and NPT (constant pressure and temperature) ensembles to stabilize the system. Three independent MD simulations, each 100 nanoseconds in duration, were run on cloud-based resources using 10 CPU cores per simulation. Post-simulation analyses, including root mean square deviation (RMSD), root mean square fluctuation (RMSF), and radius of gyration, were conducted to evaluate protein flexibility, conformational changes, and overall structural stability. 3. RESULTS 3.1. Sequence Recovery and Phylogenetic Analysis In our sequence collection from NCBI, we specifically identified one partial sequence (QSZ40499.1) out of twenty-two (amino acids and their associated gene sequences) listed in Table S1 . Employing the FASTA format, we subjected all sequences to a comprehensive analysis, utilizing various computational tools and services. This analysis covered a spectrum of aspects, including physicochemical properties, secondary and tertiary structures, functional characteristics, domains and motifs, and phylogenetic relationships. To assess evolutionary relationships, we constructed two phylogenetic dendrograms based on the alignment of amino acid sequences and their corresponding cDNA. The evolutionary distribution, as illustrated in Fig. 1 , reveals the generally comparability of the three main families of nitrogenases. The blue line signifies the second group with 18 sequences, while the dominant group, comprising 13 sequences, is denoted by red-blue lines. The third group, represented by the green line consists of a single sequence. In Fig. 1 B, the updated phylogenetic tree further shows relationships between protein sequences of different organisms and their corresponding cDNA. Three distincts groups of 22 nitrogenase cDNA sequences are depicted, containing 20, 6, and 4 sequences, respectively. Our investigation’s results reveal that the Ensifer fredii (QSZ40499.1) strain groups with AUX79023.1 and AAK53549.1 strains, demonstrating a substantial similarity of up to 98%. This cluster is further associated with strains AHL17212.1 and ABG74605.1. Notably, when comparing the relevant gene sequences of the two Ensifer fredii strains (QSZ40499.1 and AAK53549.1), we found that both strains indicated the same type of group (A. n. MT304623.1 and AF275671.1, respectively). 3.2. Physicochemical Characterization Understanding the physicochemical properties of proteins or enzymes is crucial for determining their specificity. Table 1 provides a comprehensive overview of the distinct physicochemical characteristics of nitrogenases derived from all sequences of Ensifer fredii . Table 1 Physicochemical properties of selected proteins, from different strains of Insifer fredii (Protparam server) No. Accession no. AA (aa) Mw (D) Theoretical pI Extinction coefficient (EC) Instability index (II) Aliphatic index (AI) GRAVY Subcellular localization Domain Cello PSLpred Pfam 1 QSZ40499.1 (in this study) 235 25347.98 5.12 13660 33.38 98.38 -0.109 Cytoplasmic Cytoplasm Fer4_NifH (PF00142) 2 P19068.2 296 31747.27 4.89 15150 33.98 96.96 -0.067 Cytoplasmic Cytoplasm Fer4_NifH (PF00142) 3 AUX79023.1 297 31992.50 4.85 15150 34.18 96.94 -0.104 Cytoplasmic Cytoplasm Fer4_NifH (PF00142) 4 ACE82208.1 261 28012.87 4.90 15150 32.88 96.82 -0.129 Cytoplasmic Cytoplasm Fer4_NifH (PF00142) 5 ABG74605.1 261 27995.88 4.96 15150 31.05 97.93 -0.110 Cytoplasmic Cytoplasm Fer4_NifH (PF00142) 6 ADZ23568.1 232 24942.48 5.18 13660 34.33 99.66 -0.079 Cytoplasmic Cytoplasm Fer4_NifH (PF00142) 7 ADZ23553.1 234 25156.70 5.08 13660 33.48 100.04 -0.075 Cytoplasmic Cytoplasm Fer4_NifH (PF00142) 8 ADZ23560.1 234 25188.76 5.08 34.13 34.13 98.80 -0.085 Cytoplasmic Cytoplasm Fer4_NifH (PF00142) 9 ADZ23556.1 229 24679.07 4.93 13660 35.61 95.85 -0.121 Cytoplasmic Cytoplasm Fer4_NifH (PF00142) 10 ADZ23546.1 232 24960.51 5.18 13660 34.33 97.97 -0.087 Cytoplasmic Cytoplasm Fer4_NifH (PF00142) 11 ADZ23545.1 235 25287.89 5.08 13410 33.38 99.62 -0.067 Cytoplasmic Cytoplasm Fer4_NifH (PF00142) 12 SCN47971.1 234 25156.74 5.18 13660 32.51 100.04 -0.077 Cytoplasmic Cytoplasm Fer4_NifH (PF00142) 13 ADZ23543.1 229 24665.04 4.92 13660 34.18 95.85 -0.121 Cytoplasmic Cytoplasm Fer4_NifH (PF00142) 14 ADZ23542.1 233 25073.67 5.18 13660 34.23 99.23 -0.070 Cytoplasmic Cytoplasm Fer4_NifH (PF00142) 15 ADZ23544.1 234 25188.76 5.08 13660 34.13 98.80 -0.085 Cytoplasmic Cytoplasm Fer4_NifH (PF00142) 16 ADZ23535.1 233 25057.57 5.08 13660 34.23 99.23 -0.094 Cytoplasmic Cytoplasm Fer4_NifH (PF00142) 17 ACO90391.1 246 26233.82 5.18 13660 31.54 98.37 -0.096 Cytoplasmic Cytoplasm Fer4_NifH (PF00142) 18 AAK53549.1 245 26249.85 5.00 13660 32.61 98.73 -0.099 Cytoplasmic Cytoplasm Fer4_NifH (PF00142) 19 AHL17213.1 239 25656.37 5.08 13660 32.99 102.05 -0.019 Cytoplasmic Cytoplasm Fer4_NifH (PF00142) 20 AHL17212.1 245 26513.30 4.84 15150 35.14 96.37 -0.112 Cytoplasmic Cytoplasm Fer4_NifH (PF00142) 21 ADI72792.1 236 25401.05 5.08 13660 33.28 100.85 -0.050 Cytoplasmic Cytoplasm Fer4_NifH (PF00142) 22 ABG74605.1 261 27995.88 4.96 15150 31.05 97.93 -0.110 Cytoplasmic Cytoplasm Fer4_NifH (PF00142) The molecular weights of these proteins exhibit a range from 24.66 to 31.99 KDa, with amino acid residue lengths spanning from 229 (ADZ23556.1) to 297 (AUX79023.1). Notably, all nitrogenases demonstrate acidic, as reflected in the theoretical pI values primarily falling between 4.18 and 4.8. Furthermore, the proteins exhibit high stability, indicated by instability indices consistently below 40. The aliphatic index, varying from 95.85 to 102.05 across all chosen sequences, suggests notable thermostability. The extinction coefficient, consistently light absorption at 280 nm in water, reveals a reduction in all Cys residues. Furthermore, the GRAVY values for all nitrogenase proteins range from − 0.050 to -0.129, suggesting a favorable interaction with water and classifying them as hydrophilic. A conserved motif search highlights that all proteins possess a domain belonging to the Fe4_NifH family (4Fe-4S iron-sulfur cluster proteins: PF00142), as illustrated in Figure S1 . Predictions of our protein sequence’s subcellular localization by PSLpred indicate a presence in the cytoplasmic matrix with an accuracy of 98.1%. Cello further corroborates this, predicting the protein sequence to be cytoplasmic with a reliability score of 4.711 (Table 1 ). The analysis of amino acids composition in the query sequence (Table 2 ) underscores the dominance of four residues: alanine (A), glycine (G), and glutamic acid (E), with 10.2, 9.8, 8.9, and 8.5%, respectively. 3.3. Secondary Structure Secondary structures of all nitrogenase sequences were predicted using the SOPAM tool, and percentages of alpha-helix, extended strand, beta-turn, and random coils are provided in Table 3. Among the 22 nitrogenase genes, alpha helices predominated in 16 strains, with the highest value recorded in ACO90391.1 at 42.26%. Additionally, random coils were predominant in 6 strains, ranging from 31 (P19068.2) to 35.62 (ADZ23535.1). Our query sequence (QSZ40499.1) demonstrated 34.89% amino acids in random coils, 34.47% in alpha-helices, 19.57% in extended sheets, and 11.06% in the beta-turn region (Table 3). Further insight into the query sequence's secondary structure and its graphical representation was obtained through PSIPRED (Figure S2), and this secondary analysis was extended to the remaining sequences (Figure S1). Table 2 Composition of amino acid of query sequence Table 3 Predicted secondary structure content of 22 nitrogenase proteins (SOPMA) No. Acces Alpha helix Extended stand Beta turn Random coil 1 QSZ40499.1 34.47 19.57 11.06 34.89 2 P19068.2 41.55 17.57 9.80 31 3 AUX79023.1 41.41 17.17 8.75 32.66 4 ACE82208.1 37.93 17.24 10.73 34.10 5 ABG74605.1 39.46 17.24 9.20 34.10 6 ADZ23568.1 34.91 18.97 12.07 34.05 7 ADZ23553.1 33.76 19.66 11.54 35.04 8 ADZ23560.1 32.91 20.51 12.82 33.76 9 ADZ23556.1 35.37 17.47 10.48 36.68 10 ADZ23546.1 34.48 20.69 11.21 33.62 11 ADZ23545.1 35.32 20.85 12.34 31.49 12 SCN47971.1 35.04 20.09 12.39 32.48 13 ADZ23543.1 35.81 18.34 11.35 34.50 14 ADZ23542.1 35.62 19.31 11.16 33.91 15 ADZ23544.1 32.91 20.51 12.82 33.76 16 ADZ23535.1 32.19 18.88 13.30 35.62 17 ACO90391.1 42.28 18.70 8.94 30.08 18 AAK53549.1 41.63 18.78 10.20 29.39 19 AHL17213.1 37.24 18.41 12.55 31.80 20 AHL17212.1 38.78 18.78 9.39 33.06 21 ADI72792.1 35.17 19.49 10.17 35.17 22 ABG74605.1 39.46 17.24 9.20 34.10 3.4. 3D Structure Prediction Using Homology Modeling Protein homology modeling and evaluation were systematically conducted for all five typical proteins of Ensifer fredii using the BLASTp tool [31]. Templates were chosen by identifying sequences similar to the query (QSZ40499), and phyre2, RaptorX, and Trosetta suggested the most appropriately matched template 3D protein models. Intriguingly, the AlphaFold and AlphaFold 2 server generated a model without any homolog or reference protein. In this comprehensive evaluation, four models chosen for this study (ABG74605, ACE82208, AUX79023, and sp|P19068) from the Alphafold server, exhibiting a high degree of similarity to our sequence (QSZ40499), displyed quality factor values ranging from 96.42–95.71%. Notably, our protein (QSZ40499) surpassed all, registering the highest percentage at 99.06%, thereby confirming its high-resolution structure. AlphaFold consistently produces high-quality models with excellent ERRAT scores (95.71%-99.07%) and VERIFY 3D values (~ 88%-93%). In comparison, AlphaFold 2 also performs well, but VERIFY 3D scores drop slightly (~ 70%-78%), suggesting marginally reduced model quality. Based on the Ramachandran Plot analysis, the AlphaFold-generated model exhibited the maximum number of residues in favored regions and the minimum number of residues in disallowed regions, making it more acceptable than the other models (Table 4). Table 4 3D Modeling score of predicted models of Nitrogenase enzyme using multiple server Server No PROVE (Outlier) Z score ERRAT VERIFY 3D Ramashadran PLOT Mean Quality Factor 3D-ID Score Most Favored regions Additional allowed region (%) Generally allowed region (%) Disallowed region (%) AlphaFold ABG74605 3,2% (35) 0,519 96.4286 88.85% 0,957 0,043 0 0 ACE82208 3,9% (37) 0,538 97.9839 89.66% 0,942 0,058 0 0 AUX79023 3,2% (35) 1 98.2394 86.15% 0,946 0,054 0 0 QSZ40499 3,7% (27) 0,563 99.0698 93.19% 0,966 0,034 0 0 sp|P19068 3,2% (35) 0,526 95.7143 88.51% 0,957 0,043 0 0 AlphaFold 2 ABG74605 98.8048 78.54% 0,924 0,076 0 0 ACE82208 98.4064 78.16% 0,929 0,067 0,004 0 AUX79023 97.5610 78.11% 0,934 0,062 0,004 0 QSZ40499 98.2143 70.21% 0,931 0,069 0 0 sp|P19068 97.5524 77.70% 0,938 0,058 0,004 0 Phyre 2 ABG74605 87.8136 88.89% 0,876 0,116 0,008 0 ACE82208 84.1897 93.49% 0,866 0,121 0,013 0 AUX79023 86.2454 90.97% 0,891 0,097 0,008 0,004 QSZ40499 91.9643 89.36% 0,862 0,123 0,015 0 sp|P19068 87.8136 88.89% 0,876 0,116 0,008 0 RapptorX ABG74605 11,5% (102) 0,877 85.7708 90.80% 0,906 0,085 0,009 0 ACE82208 9,9% (89) 0,723 92.8854 92.72% 0,911 0,08 0,009 0 AUX79023 11,7% (103) 0,791 88.5375 94.25% 0,888 0,085 0,022 0,004 QSZ40499 92.3767 83.40% 0,837 0,138 0,015 0,01 sp|P19068 11,1% (115) 0,761 90.9091 90.20% 0,864 0,113 0,019 0,004 TRosetta ABG74605 5,3% (48) 0,131 90.9091 95.79% 0,929 0,067 0,004 0 ACE82208 6,4% (57) 0,142 94.0476 96.17% 0,929 0,062 0,009 0 AUX79023 5,8% (60) 0,06 81.9444 91.22% 0,911 0,074 0,016 0 QSZ40499 6,3% (45) 0,172 89.2377 95.74% 0,936 0,054 0,01 0 sp|P19068 6% (62) 0,078 85.6115 86.82% 0,922 0,074 0 0,004 The selected model (AlphaFold) showcased residues in favored regions ranging from 94.2% (ACE82208) to 96.6% (QSZ40499), with corresponding percentages in allowed regions ranging from 3.4% (QSZ40499) to 5.8% (ACE82208). No residues were identified in the disallowed regions across all sequences, with the overall quality factor of ERRAT values ranging between 95.6% and 99.06% (Table 4). 3.5. Refinement Structure As a crucial step to refine the precision of initial structures and rectify local inaccuracies, the predicted 3D structure underwent scrutiny. The refined structure from the AlphaFold server was subject to quality assessments through ERRAT and Ramachandran plots. The revised structure's Ramachandran plot demonstrated an absence of residues in forbidden regions (Table 5, Figure S4, S6). Notably, the 3Drefine server emerged as the optimal choice for our query, evident in Z scores ranging from 0.057 to 0.277 and ERRAT (Overall Quality Factor) values between 98.8% and 100% (Table 5). This rigorous refinement process enhances the reliability of the structural insights gained from the study. Table 5 Refinement score of predicted models of Nitrogenase enzyme using multiple server Server No PROVE (Outlier) Z score ERRAT (Overall Quality Factor) Ramashadran PLOT Mean Quality Factor 3D-ID Score Most Favored regions (%) Additional allowed region (%) Generally allowed region (%) Disallowed region (%) 3Drefine ABG74605 0,025 0,057 100 88.85% 94,6 5,4 0 0 ACE82208 0,037 0,277 98,8 97.32% 93,3 6,7 0 0 AUX79023 3,6 0,28 98,95 89.86% 93,8 6,2 0 0 QSZ40499 3 0,224 99,55 96.17% 95,6 4,4 0 0 sp|P19068 2,6 0,251 99,29 88.85% 95,3 4,7 0 0 Deeprefiner ABG74605 0,028 0,442 95 89.53% 93,8 6,2 0 0 ACE82208 0,032 0,43 97,16 98.47% 91,1 8,9 0 0 AUX79023 2,50% 0,416 96,099 92.91% 91,4 8,6 0 0 QSZ40499 3,1 0,48 100 85.53% 91,6 8,4 0 0 sp|P19068 3,4 0,43 98,58 89.19% 94,6 5,4 0 0 Galaxy ABG74605 98,93 88.18% 96,9 3,1 0 0 ACE82208 0,047 0,59 98,78 92.72% 95,5 4,5 0 0 AUX79023 95,75 89.86% 97,3 2,7 0 0 QSZ40499 5 0,579 98,61 90.21% 97,5 2,5 0 0 sp|P19068 95,39 89.19% 97,7 2,3 0 0 3.6 Molecular Dynamics Simulation Structural Stability and Flexibility The molecular dynamics (MD) simulations provided valuable insights into the structural stability of the nitrogenase Fe protein from Ensifer fredii . The Solvent Accessible Surface Area (SASA) (Fig. 2A) exhibited moderate fluctuations throughout the simulation, reflecting the protein’s conformational flexibility while maintaining overall structural integrity. The trend in SASA suggests that the protein stayed within a defined range of solvent exposure, indicating reasonable stability. The temperature profile (Fig. 2B) remained stable, with only minor fluctuations, suggesting that the system was well-equilibrated. Additionally, the root mean square deviation (RMSD) showed that the protein conformation reached a stable plateau after an initial rise, confirming that the system achieved equilibration. Minor fluctuations around the plateau reflect routine conformational adjustments without compromising structural integrity. Protein Flexibility and Conformational Dynamics The root mean square fluctuation (RMSF) analysis (Fig. 2D) revealed regions of the protein with higher flexibility. These regions may correspond to active sites or other functionally relevant areas of the protein that undergo conformational changes during nitrogenase activity. When RMSF was compared with SASA data, regions displaying high flexibility also demonstrated increased solvent exposure (Fig. 2C), indicating that flexible areas of the protein may be more accessible for functional interactions. Compactness and Thermodynamic Stability The radius of gyration (Rg) plot (Fig. 2G) showed that the overall compactness of the protein was maintained, with the Rg remaining relatively constant throughout the simulation. Although some anisotropy was observed in the dimensions Rgx, Rgy, and Rgz, these fluctuations were minor and did not significantly impact the structural integrity. The potential energy profile also plateaued after a period of equilibration, confirming that the protein achieved a stable energy state (Figs. 2E and 2F). The overall energy conservation during the MD simulation was reflected in the Gromacs energy plot (Fig. 2H), suggesting the system remained thermodynamically stable. 4. DISCUSSION Nitrogenase serves a crucial role in converting atmospheric nitrogen dioxide to ammonia (NH3). However, the Mo-Fe-S complex nitrogenase enzyme is permanently inactivated by O2. To counter this, free-living diazotrophs employ respiratory protection to lower internal oxygen concentration, while plant polysaccharides in bacteroids maintain rapid aerobic respiratory turnover to support nitrogenase activity, potentially producing reactive oxygen species (ROS) as byproducts. Notably, three distinct nitrogen-fixing systems—Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase—have been identified, each composed of a two-component complex metalloenzyme system with dinitrogenase reductase as an iron protein and dinitrogenase as a metal cofactor [ 32 ]. In the quest for a deeper understanding of nitrogenase, in silico protein modeling has emerged as a cost-effective and expeditious alternative to experimental approaches [ 33 ]. Computational tools now play an increasingly pivotal role in navigating sequence space and enhancing laboratory evolution efficiency. Additionally, Sefdi et al. asserted the efficacy of bioinformatics tools as an effective bridge connecting protein sequences to their 3D structures. 4.1. Physiochemical Characterization A computationally based investigation into the physicochemical behavior of nitrogenases from several Ensifer spp. was performed, and this analysis provided a theoretical insight into protein nature. The pH at which a protein attains neutrality, referred to as the isoelectric or isoionic point [ 35 ], was determined across 22 strains, yielding scores ranging from 4.84 to 5.18. This range suggests a moderate acidity in naturally occurring nitrogenases. The stability of proteins, measured by the instability index (II), designates values exceeding 40 as indicative of instability [ 36 ]. Interestingly, all nitrogenase strains of E. fredii exhibited instability scores below 40, signifying the stability of this protein. According to IKAI (1980), the aliphatic index (AI), influencing a protein's thermal stability, gauges the proportion of a protein's relative volume occupied by aliphatic amino acids in the side chain. Demonstrating exceptional thermostability, all enzymes exhibited an AI value exceeding 95.85%. Ensifer fredii , among the limited bacteria displaying such characteristics, showcased the highest concentrations of alanine (10.2%), glycine (9.8%), and leucine (8.9%). This composition likely contributed to the heightened thermostability observed in E. fredii . The enzymes were confirmed as thermostable, characterized by elevated aliphatic index values and a substantial percentage of alpha-helix structures. The interplay between water and protein was elucidated through the GRAVY metric. A positive GRAVY value denotes hydrophobicity, while a negative value signifies enhanced hydrophilic interactions with water. In addition, the polar characteristics of the hydrophilic amino acids glycine, glutamic acid, lysine, aspartic acid, and serine make them more likely to interact in aqueous environments. Cystidine residues are crucial in creating disulfide connections between different protein components. Disulfide bonds are crucial for protein folding and stabilization of the unfolded state by reducing entropy. However, in most cases, disulfide bridges are lacking due to cysteine residues' extremely low occurrence [ 38 ]. 4.2. Secondary Structural Analysis The prediction of secondary protein structures from sequences serves as a crucial link connecting primary and tertiary structure predictions [ 39 ]. Random coil (34.89%) and alpha helix (34.47%) were the two most common secondary structures, while extended stand (19.57%) and beta-turn (11.06%) were the other configurations [Figure S3]. High scores in random coils signify the absence of regular secondary structures, providing flexibility for conformational changes, such as enzyme turnover [ 40 ]. Meanwhile, extended strands and alpha-helices contribute to protein stability. In a parallel investigation by Satyanarayana et al. Nif-A sequences in Bradyrhizobium japonicum , Rhizobium leguminosarum , and Misorhizobium ciceri showcased dominance in random coil, extended strand, and beta-twist configurations during secondary structure analysis. This reinforcing the consistency of our findings [ 39 ]. The oxygen sensitivity of NifH proteins appears to correlate with the presence of alanine and glycine residues, indicating a potantiel connection to the coordination of metal ions and the redox status of the proteins [ 42 ]. Employing in silico protein structure research emerges as a valuable method for exploring the structural and functional characteristics of proteins [ 43 ]. Acquiring twenty-two sequences (both amino acids and their corresponding gene sequences) from NCBI, our phylogenetic analysis of cDNA for nitrogenase proteins revealed a congruent clustering with the protein comparison (Figure S3). The phylogenetic tree, indicating two distinct groups within the selected strains. This result aligns with the approaches of other researchers [ 39 , 40 ], who used a similar method to assess associations between taxa protein sequences and their corresponding cDNA sequences. Notably, the high reliability of the tree is underscored by bootstrap values ranging from 98–100%. 4.3. Tertiary Structure Analysis Confirming information derived from NMR/X-ray crystallography-based approaches poses a significant challenge in predicting a protein's three-dimensional (3D) model through in silico analysis [ 44 ]. Due to the absence of experimental data in the protein data bank, tertiary structure prediction homology modeling was employed to predict the 3D structure of the nitrogenase protein. RaptorX, TrRosetta, and Phyre2 created a structural model by choosing the optimal template for modeling, whereas AlphaFold created a protein model based solely on the amino acid structure without a template or homologous structure. Ensifer fredii was chosen as a representative species for homology protein modeling and submission, clarifying the nitrogenase protein structure of Ensifer spp. The selection was based on Z score, an overall quality factor from the SAVES server, and the Ramachandran plot. Homology modeling was conducted for all five proteins (see figures S3 and S5, Table 4 ). Phyre2's alignment file was adjusted to accommodate the overlap between the secondary structure elements of the template and the predicted secondary structure profile of the sequence. On the other hand, TrRosetta and RaptorX utilized random generation and spatial resistance to produce five initial protein models. The quality of the model was assessed using the Ramachandran plot, where more than 90% of residues were located in the preferred regions, indicating high quality [ 46 ]. Examining the PDB structure of Ensifer fredii (QSZ40499) protein, the Ramachandran plot revealed that 97.9% of residues occupied the most preferred region. A high-quality model was further indicated by a Z-score falling within the range of 0–1 [ 47 ] and 1 [ 48 ] compared to a non-redundant PDB structure set. Computational methods were employed to examine and analyze the tertiary structures of the selected nitrogenase enzymes. Quality parameters including ERRAT, Z-score, and the Ramachandran plot were utilized. A high-resolution structure, with an overall quality factor of 95% or higher, was considered good [ 48 ]. The retrieved Z-score mean and standard deviation from the SAVES server were 0.524 and 0.538, respectively, aligning closely with the expectation for a high-resolution structure (Table 5 ) [ 47 ]. three homology modeling programs (Physical2, Trosetta, and RaptorX) were employed, along with the template-free approach of AlphaFold and AlphaFold 2. The results consistently affirmed the reliability and precision of the predicted structure by AlphaFold. Although our model includes conserved nucleotide-binding motifs, the absence of explicit ATP/ADP coordinates in the AlphaFold structure may limit direct visualization of the nucleotide-binding conformation. Future studies incorporating ligand-docking simulations could address this gap. Although our model includes several critical residues for nucleotide binding, it lacks the critical N-terminal sequence stretch required for this binding. We believe that this constraint will not jeopardize the current investigation. 4.4. Model Refinement Refining the projected 3D protein model is crucial to enhance the prediction models' quality. Three servers—3Drefiner, Galaxy, and Deeprefiner—were employed for this purpose, aiming to advance the experimental accuracy of models for subsequent computational investigations [ 33 ]. After analyzing the Ramachandran plot for the PDB structure of selected queries. The Galaxy server was chosen due to the high percentage range (95,5–97,7%) of residues present in the most favored region. The 3Drefine server appears to be the best server of refinement to our query based on a Z score between (0,057-0.277) and ERRAT (Overall Quality Factor) between (98.8–100) (Table 5 , Figure S4). The increased overall model quality factor obtained through ERRAT analysis for our query (QSZ40499) indicated structural improvement, rendering it suitable for additional docking studies (presence of more than 90% residues in the favored (red) region of the Ramachandran plot [ 46 ]. The model's accession number is PM0084218, and it was uploaded to the Protein Model Database (PMDB) database in pdb format after being refined and reviewed ( https://bioinformatics.cineca.it/PMDB/index.php ). Protein interactions with ligands, ions, and other proteins play a crucial role in determining protein activity. However, the focus on protein structure is fundamental as the initial step for computational functional investigations [ 42 ]. For an improved 3D model, considerations of oligomeric states and protein-ligand complexes are essential, reflecting real-world protein interactions with ligands such as ions, inhibitors, and peptides. Neglecting these intricate molecular systems could result in the refinement of artificial protein models. 4.5 Molecular Dynamics Simulation The MD simulation results reveal that the nitrogenase Fe protein exhibits a delicate balance between structural stability and functional flexibility. The moderate SASA fluctuations suggest that while the protein remains solvent-exposed in certain regions, its core structure remains robust. This solvent exposure, particularly in regions with higher RMSF values, may be critical for the protein's biological function, enabling the necessary conformational changes required during nitrogen fixation [ 49 ]. The RMSD data further support the notion of a stable conformation after the initial equilibration phase, with only minor fluctuations indicative of routine conformational sampling [ 50 ]. The plateauing of the RMSD and potential energy highlights that the system has reached a stable energetic and structural state [ 51 ]. The flexibility observed in the RMSF data corresponds to regions that might be involved in dynamic interactions, such as substrate binding or catalysis [ 52 ]. These flexible, solvent-exposed regions could play a critical role in the protein's functional mechanisms. The combination of high flexibility and solvent exposure often corresponds to dynamic functional regions, which are essential for the enzymatic activity of nitrogenase. The constant radius of gyration suggests that the overall compactness of the protein is preserved, indicating that the core structure remains stable. This compactness, despite minor anisotropic variations, aligns with the energy conservation observed throughout the simulation, underscoring the protein’s ability to remain structurally intact while maintaining flexibility where needed. The molecular dynamics simulations reveal that the [Fe-S] cluster plays a pivotal role in stabilizing the nitrogenase Fe protein’s core, as indicated by RMSF values below 1.5 Å in the cluster region (Fig. 2 D). This stability, driven by the cluster’s coordination, likely supports its function in electron transfer during nitrogen fixation, particularly under redox stress. Meanwhile, the flexibility of solvent-exposed loops (RMSF > 3 Å) suggests these regions contribute to the protein’s adaptability, potentially aiding interactions critical for activity. This interplay between a rigid core and dynamic periphery underscores the structural balance essential for nitrogenase function. The MD simulations demonstrate that the nitrogenase Fe protein from Ensifer fredii can maintain a stable structure with the necessary flexibility for functional activity. These properties likely contribute to its role in nitrogen fixation, where dynamic interactions and conformational adjustments are crucial for optimal performance. Future experimental studies may focus on further exploring these flexible regions to better understand their role in nitrogenase function. 5. CONCLUSION This study provides significant in silico insights into the nitrogenase enzyme of E. fredii , emphasizing its physicochemical and structural properties. Key findings include sequence lengths ranging from 229–297 amino acids across the analyzed NifH proteins, with our primary focus on QSZ40499 (residues 32–266), demonstrating a predominance of hydrophilic residues (34.89% random coils) and dynamic structural behavior essential. AlphaFold modeling, validated by Z-scores and ERRAT metrics, demonstrated the enzyme’s balance between stability and flexibility, essential for nitrogen fixation. Molecular dynamics simulations revealed solvent-exposed flexible regions facilitating functional activity. These findings establish a foundation for future experimental studies and biotechnological applications aimed at enhancing nitrogen fixation for agricultural and industrial purposes. Future studies should prioritize experimental validation of the nitrogenase enzyme’s catalytic properties. Additionally, integrating structural insights with biotechnological strategies can optimize nitrogen fixation for sustainable agriculture and industry. Declarations Author contributions The conceptualization of the study was proposed by Mohamed Hnini and Karim Rabeh, both of whom made equal contributions to result analysis and manuscript writing. All authors have reviewed and approved the final manuscript. Funding There is no funding to report for this study. Data Availability All data generated or analyzed during this study are included in this published article as tables or figures. Conflicts of Interest The authors declare no competing interests. Ethical Approvals Not applicable. Informed Consent Not applicable. References Burris, R.H.; Roberts, G.P. Biological Nitrogen Fixation. Annu. Rev. Nutr. 1993 , 13 , 317–335, doi:10.1146/annurev.nu.13.070193.001533. Raymond, J.; Siefert, J.L.; Staples, C.R.; Blankenship, R.E. The Natural History of Nitrogen Fixation. Mol. Biol. Evol. 2004 , 21 , 541–554, doi:10.1093/molbev/msh047. Burgess, B.K.; Lowe, D.J. Mechanism of Molybdenum Nitrogenase. Chem. 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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-5209284","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":436508302,"identity":"566d2400-a558-47b4-8977-86b358b08213","order_by":0,"name":"Mohamed Hnini","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYBACAwbGBoYEA4n6fhAvoYBoLRU2jDMbQFoMiNICAmfSGDccQHDxA3Pp5rYHD9sOMxufX5344YEBgzy/2AH8WiznHGw3SGw7zGZ24+1mCaDDDGfOTiDgsBuJbRJALTxmN85uAGlJMLhNpBYJ4xlnN/8gXkvCmTQDA/7ebSTYAgzkBIkbvNssgBFEjF/Sn0n+MJBI4O8/u/nmjwobeX5pAloQQAKsUoJY5SDAf4AU1aNgFIyCUTCSAADJyUiomLme7QAAAABJRU5ErkJggg==","orcid":"","institution":"High School of Technology Laayoune, , Ibn Zohr University","correspondingAuthor":true,"prefix":"","firstName":"Mohamed","middleName":"","lastName":"Hnini","suffix":""},{"id":436508303,"identity":"b493ac21-691a-4e53-8c9d-4fafa8bc9724","order_by":1,"name":"Karim Rabeh","email":"","orcid":"","institution":"National Institute of Agricultural Research","correspondingAuthor":false,"prefix":"","firstName":"Karim","middleName":"","lastName":"Rabeh","suffix":""}],"badges":[],"createdAt":"2024-10-05 14:08:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5209284/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5209284/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79687504,"identity":"a3449156-42e1-4406-be52-614c82d6b5c0","added_by":"auto","created_at":"2025-04-01 13:56:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":300302,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of 22 different bacterial Nitogenases by Maximum Likehood using MEGAX. A) Phylogenetic analysis of proteins from 22 different strains of \u003cem\u003eEnsifer fredii \u003c/em\u003eB) Phylogenetic analysis of cDNA from 22 different strains of \u003cem\u003eEnsifer fredii\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5209284/v1/b6bb20c04eca2a6d2700344c.png"},{"id":79687505,"identity":"5d60d13c-dad8-467a-9534-b32ea6ff6eb4","added_by":"auto","created_at":"2025-04-01 13:56:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":159961,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Solvent Accessible Surface Area (SASA) analysis of the NifH protein (QSZ40499) during the molecular dynamics simulation, highlighting changes in solvent exposure over time. (B) Energy profile of the system during the initial equilibration phase of the molecular dynamics simulation, showing stability achieved using GROMACS. (C) Area per residue of the NifH protein (QSZ40499), illustrating solvent exposure and variability across different regions of the protein structure. (D) Root Mean Square Fluctuation (RMSF) of the NifH protein (QSZ40499), indicating residue-specific flexibility throughout the simulation. (E) Root Mean Square Deviation (RMSD) of the NifH protein (QSZ40499) backbone during the simulation, reflecting structural stability over time. (F) Potential energy profile of the NifH protein (QSZ40499), demonstrating system stabilization and energy minimization over the molecular dynamics simulation. (G) Energies of the NifH protein (QSZ40499) during the simulation, illustrating various energy components over time.(H) Radius of gyration (Rg) of the NifH protein (QSZ40499), illustrating protein compactness and structural integrity throughout the simulation.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5209284/v1/631a1961f1559f4055e9518d.png"},{"id":79689271,"identity":"f7556e84-ea7b-48a8-a5e5-e1d6ac1e9936","added_by":"auto","created_at":"2025-04-01 14:20:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2146256,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5209284/v1/e62b1d17-0c64-4711-8adc-76849e4159bc.pdf"},{"id":79687507,"identity":"dd31c0f2-f00b-480d-8d93-5a0bb42f9b64","added_by":"auto","created_at":"2025-04-01 13:56:21","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2439857,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5209284/v1/b99f4039ee54f369ee7059e4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"In Silico Exploration of Nitrogenase in Ensifer fredii Unraveling Structural Homology Functional and Phylogenetic Relationships and Molecular Dynamics ","fulltext":[{"header":"Article Highlights","content":"\u003cul class=\"decimal_type\"\u003e\n \u003cli\u003eThis study analyzes the Nitrogenase sequence gene (QSZ40499) from \u003cem\u003eEnsifer fredii\u003c/em\u003e, emphasizing stability and functionality.\u003c/li\u003e\n \u003cli\u003e22 NifH proteins undergo a thorough examination of primary, secondary, and tertiary structures using diverse biocomputational tools.\u003c/li\u003e\n \u003cli\u003eQSZ40499 demonstrates high stability, hydrophilicity, and cytoplasmic localization, with a molecular weight of 25.34 KD.\u003c/li\u003e\n \u003cli\u003eSecondary structure analysis reveals a predominant random coil (34.89%) and alpha helix (34.47%) in the NifH protein.\u003c/li\u003e\n \u003cli\u003eAlphafold is a reliable tool for NifH protein modeling, and the 3Drefiner server is suitable for subsequent refinement, contributing valuable insights to bioinformatics research.\u003c/li\u003e\n \u003cli\u003eMolecular dynamics simulations show that the nitrogenase Fe protein maintains structural stability while possessing essential flexibility for its functional role in nitrogen fixation.\u003c/li\u003e\n\u003c/ul\u003e\n"},{"header":"1. INTRODUCTION","content":"\u003cp\u003eThe primary source of fixed nitrogen (N) in the global biogeochemical cycle is biological conversion to ammonia (NH), facilitated by the microbial enzyme Nitrogenase [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Three types of nitrogenase enzymes have been identified: Molybdenum (Mo)-dependent, vanadium (V)-dependent, and iron (Fe)-dependent [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These enzymes are encoded by distinct gene clusters known as \u003cem\u003enif\u003c/em\u003e, \u003cem\u003evnf\u003c/em\u003e, and \u003cem\u003eanf\u003c/em\u003e [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The extensively studied Mo-nitrogenase studied variant comprises two components: Fe proteins encoded by \u003cem\u003enifH\u003c/em\u003e genes and MoFe proteins encoded by \u003cem\u003enifD\u003c/em\u003e and \u003cem\u003enifK\u003c/em\u003e genes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eProtein structure modeling serves as a method to comprehend biological processes at the molecular level. While structural information on NifH proteins (EC 1.18.6.1, nitrogenase component II)) is known for some bacteria, such as \u003cem\u003eAzotobacter\u003c/em\u003e, \u003cem\u003eClostridium\u003c/em\u003e, and \u003cem\u003eArthrobacter sp.\u003c/em\u003e[\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], it is lacking for \u003cem\u003eEnsifer\u003c/em\u003e sp. This can be explained by two factors: the lack of X-ray crystallographic research on the nitrogenase iron protein produced by \u003cem\u003eEnsifer\u003c/em\u003e sp. and the absence of reliable three-dimensional (3D) modeling of the NifH protein.\u003c/p\u003e \u003cp\u003eUnderstanding a protein's function necessitates knowledge of its 3D structure. Experimental determination through X-ray crystallography or NMR spectroscopy proves challenging, complicated, expensive, and time-consuming [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Addressing this, computational approaches, or alternative methods, particularly homology modeling, have gained widespread acceptance for predicting \u003cem\u003ein silico\u003c/em\u003e 3D protein structures [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study uses various biocomputational methods to characterize the enzyme Nitrogenase from \u003cem\u003eEnsifer\u003c/em\u003e sp., an understudied subject. The investigation explores the structural, physicochemical, phylogenetic, and functional aspects of the nifH protein from \u003cem\u003eEnsifer fredii\u003c/em\u003e. This specific strain was isolated from the nodules of \u003cem\u003eVachellia tortilis\u003c/em\u003e subsp. \u003cem\u003eraddiana\u003c/em\u003e, a resilient leguminous tree thriving naturally in a challenging arid climate.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn silico\u003c/em\u003e protein analysis is gaining popularity among scientists due to its significant contribution to obtaining baseline data in a timely and appropriate manner. This study will aid researchers in gaining a foundational understanding of this crucial enzyme, a member of the \u003cem\u003eRhizobiaceae\u003c/em\u003e family, and explores additional research directions, such as building a complex protein docking despite the complexity of symbiotic processes and nitrogen fixation mechanisms.\u003c/p\u003e \u003cp\u003eThis study is guided by the following research questions:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eWhat are the key structural and functional features of the nitrogenase Fe protein (NifH) in \u003cem\u003eEnsifer fredii\u003c/em\u003e as determined through in silico methods?\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eHow does the structure of the NifH protein from \u003cem\u003eEnsifer fredii\u003c/em\u003e compare to other homologous proteins across bacterial species in terms of phylogenetics and functionality?\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eWhat insights can molecular dynamics simulations provide about the stability and flexibility of the NifH protein during nitrogen fixation?\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eBased on these questions, the study hypothesizes that the nitrogenase Fe protein (NifH) in \u003cem\u003eEnsifer fredii\u003c/em\u003e, modeled using advanced \u003cem\u003ein silico\u003c/em\u003e approaches, demonstrates a unique combination of structural stability and functional flexibility essential for efficient nitrogen fixation. Specifically, the structural stability ensures the protein maintains its conformational integrity under physiological conditions, while the functional flexibility enables dynamic conformational changes necessary for its catalytic role in converting atmospheric nitrogen to ammonia. This balance between stability and flexibility is likely conserved across homologous proteins, reflecting an optimized functional approach for nitrogen fixation in diverse bacterial species.\u003c/p\u003e"},{"header":"2. MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Sequence Retrieval, Alignment, and Phylogenetic Analysis\u003c/h2\u003e \u003cp\u003eThe nitrogenase Fe protein, derived from the 'NifH' gene in \u003cem\u003eEnsifer fredii\u003c/em\u003e, was isolated from the root nodule of \u003cem\u003eVachellia tortilis\u003c/em\u003e subsp. \u003cem\u003eraddiana\u003c/em\u003e (QSZ40499). Using the NCBI database, the NifH amino acid partial sequence (accession no. QSZ40499.1) was obtained in FASTA format and employed as a BLAST query against a nonredundant protein database. For subsequent analysis, a subset of 22 sequences with an E-value below 4.30e-173 was selected. Multiple sequence alignments were performed using Clustal Omega (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/jdispatcher/msa/clustalo\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/jdispatcher/msa/clustalo\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), encompassing deduced amino acid and cDNA sequences. The construction of a phylogenetic tree using MEGAX [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] through the maximum likelihood technique, supported by one thousand bootstrap replicates, resulted in two distinct trees representing amino acid sequences and cDNA from various \u003cem\u003eEnsifer fredii\u003c/em\u003e nitrogenase strains.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Physicochemical Characterization\u003c/h2\u003e \u003cp\u003eThe ExPaSy ProtParam tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) facilitated the determination of physicochemical characteristics for nitrogenase Fe protein sequences. These parameters included the grand average of hydropathogenicities (GRAVY), molecular weight (Mw), isoelectric point (pI), extinction coefficient (EC), instability index (II), and aliphatic index (AI).\u003c/p\u003e \u003cp\u003ePrediction of the family domain for each protein was executed using ScanProsite (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://prosite.expasy.org/scanprosite/\u003c/span\u003e\u003cspan address=\"http://prosite.expasy.org/scanprosite/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), PFAM (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam.xfam.org\u003c/span\u003e\u003cspan address=\"http://pfam.xfam.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the MOTIF search (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genome.jp/tools/motif\u003c/span\u003e\u003cspan address=\"https://www.genome.jp/tools/motif\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Subcellular structures were localized through Cello (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cello.life.nctu.edu.tw\u003c/span\u003e\u003cspan address=\"http://cello.life.nctu.edu.tw\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and PSLpred [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Structure Prediction and Evaluation\u003c/h2\u003e \u003cp\u003eThe secondary structure of amino acid sequences was predicted by SOPMA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://npsa-prabi.ib,cp.fr/cgi\u003c/span\u003e\u003cspan address=\"https://npsa-prabi.ib,cp.fr/cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e bin/npsa_automat.pl?page\u0026thinsp;=\u0026thinsp;npsa_sopma.html) and PSIPRED (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinf.cs.ucl.ac.uk/psipred/\u003c/span\u003e\u003cspan address=\"http://bioinf.cs.ucl.ac.uk/psipred/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs the 3D model template for our query sequence was unavailable in the Protein Data Bank (PDB), PHYRE2 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], trRosetta (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://yanglab.nankai.edu.cn/trRosetta/\u003c/span\u003e\u003cspan address=\"https://yanglab.nankai.edu.cn/trRosetta/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], RaptorX (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://raptorx.uchicago.edu/\u003c/span\u003e\u003cspan address=\"http://raptorx.uchicago.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], AlphaFold and AlphaFold II were utilized to construct homology 3D structures of five nitrogenase proteins, including our protein (Accession no. QSZ40499.1). Visualization of the model was conducted using Discovery Studio, version 21.1.0.[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEvaluation and verification of the predicted protein model of \u003cem\u003eEnsifer fredii\u003c/em\u003e Nitrogenase was performed using the SAVES (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://servicesn.mbi.ucla.edu/SAVES/\u003c/span\u003e\u003cspan address=\"https://servicesn.mbi.ucla.edu/SAVES/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) server. This involved various tools, such as the Ramachandran plot generated by the RAMPAGE server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mordred.bioc.cam.ac.uk/rapper/rampage.php\u003c/span\u003e\u003cspan address=\"http://mordred.bioc.cam.ac.uk/rapper/rampage.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], Verify3D [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and ERRAT for the examination of crystallographic structures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Model Refinement\u003c/h2\u003e \u003cp\u003eTo refine the predicted 3D protein model, three servers were utilized: 3Drefine protein structure refinement server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://sysbio.rnet.missouri.edu/3Drefine\u003c/span\u003e\u003cspan address=\"http://sysbio.rnet.missouri.edu/3Drefine\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], DeepRefiner protein structure refinement server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://watson.cse.eng.auburn.edu/DeepRefiner\u003c/span\u003e\u003cspan address=\"http://watson.cse.eng.auburn.edu/DeepRefiner\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and GalaxyWEB web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://galaxy.seoklab.org\u003c/span\u003e\u003cspan address=\"http://galaxy.seoklab.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The refined nitrogenase protein models underwent evaluation and verification using the SAVES server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://services.mbi.ucla.edu/SAVES/\u003c/span\u003e\u003cspan address=\"http://services.mbi.ucla.edu/SAVES/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which included assessments through the Ramachandran plot, verify3D and ERRAT.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Molecular Dynamics Simulation\u003c/h2\u003e \u003cp\u003eTo further assess the stability and behavior of the refined nitrogenase Fe protein model, a molecular dynamics (MD) simulation was performed using GROMACS, facilitated by the Visual Dynamics platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://visualdynamics.fiocruz.br/\u003c/span\u003e\u003cspan address=\"https://visualdynamics.fiocruz.br/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The simulation was executed in an APO state, employing the AMBER99SB-ILDN force field to ensure accurate modeling of protein dynamics. The system was solvated in a TIP3P water box, maintaining a 1.0 nm buffer from the protein to the box boundaries, and neutralized with counterions.\u003c/p\u003e \u003cp\u003eThe simulation environment was adjusted to mimic biologically relevant conditions:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eTemperature: Maintained at 300 K using the Nose-Hoover thermostat.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003ePressure: Maintained at 1 bar using the Parrinello-Rahman barostat.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eThe simulation was conducted with a timestep of 2 femtoseconds (fs), following standard GROMACS protocols. Prior to the production run, energy minimization was performed to eliminate steric clashes, followed by equilibration in NVT (constant volume and temperature) and NPT (constant pressure and temperature) ensembles to stabilize the system.\u003c/p\u003e \u003cp\u003eThree independent MD simulations, each 100 nanoseconds in duration, were run on cloud-based resources using 10 CPU cores per simulation. Post-simulation analyses, including root mean square deviation (RMSD), root mean square fluctuation (RMSF), and radius of gyration, were conducted to evaluate protein flexibility, conformational changes, and overall structural stability.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Sequence Recovery and Phylogenetic Analysis\u003c/h2\u003e \u003cp\u003eIn our sequence collection from NCBI, we specifically identified one partial sequence (QSZ40499.1) out of twenty-two (amino acids and their associated gene sequences) listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Employing the FASTA format, we subjected all sequences to a comprehensive analysis, utilizing various computational tools and services. This analysis covered a spectrum of aspects, including physicochemical properties, secondary and tertiary structures, functional characteristics, domains and motifs, and phylogenetic relationships.\u003c/p\u003e \u003cp\u003eTo assess evolutionary relationships, we constructed two phylogenetic dendrograms based on the alignment of amino acid sequences and their corresponding cDNA. The evolutionary distribution, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, reveals the generally comparability of the three main families of nitrogenases. The blue line signifies the second group with 18 sequences, while the dominant group, comprising 13 sequences, is denoted by red-blue lines. The third group, represented by the green line consists of a single sequence. In Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, the updated phylogenetic tree further shows relationships between protein sequences of different organisms and their corresponding cDNA. Three distincts groups of 22 nitrogenase cDNA sequences are depicted, containing 20, 6, and 4 sequences, respectively. Our investigation\u0026rsquo;s results reveal that the \u003cem\u003eEnsifer fredii\u003c/em\u003e (QSZ40499.1) strain groups with AUX79023.1 and AAK53549.1 strains, demonstrating a substantial similarity of up to 98%. This cluster is further associated with strains AHL17212.1 and ABG74605.1. Notably, when comparing the relevant gene sequences of the two \u003cem\u003eEnsifer fredii\u003c/em\u003e strains (QSZ40499.1 and AAK53549.1), we found that both strains indicated the same type of group (A. n. MT304623.1 and AF275671.1, respectively).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Physicochemical Characterization\u003c/h2\u003e \u003cp\u003eUnderstanding the physicochemical properties of proteins or enzymes is crucial for determining their specificity. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e provides a comprehensive overview of the distinct physicochemical characteristics of nitrogenases derived from all sequences of \u003cem\u003eEnsifer fredii\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysicochemical properties of selected proteins, from different strains of \u003cem\u003eInsifer fredii\u003c/em\u003e (Protparam server)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAccession no.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAA\u003c/p\u003e \u003cp\u003e(aa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMw (D)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTheoretical pI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eExtinction coefficient (EC)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eInstability index (II)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAliphatic index (AI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGRAVY\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003eSubcellular localization\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eDomain\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCello\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePSLpred\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003ePfam\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQSZ40499.1 (in this study)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e235\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25347.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e33.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e98.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCytoplasmic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eFer4_NifH\u003c/b\u003e\u0026nbsp;\u003cb\u003e(PF00142)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP19068.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e296\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31747.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e33.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e96.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.067\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCytoplasmic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eFer4_NifH\u003c/b\u003e\u0026nbsp;\u003cb\u003e(PF00142)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAUX79023.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e297\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31992.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e34.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e96.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCytoplasmic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eFer4_NifH\u003c/b\u003e\u0026nbsp;\u003cb\u003e(PF00142)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eACE82208.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e28012.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e32.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e96.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.129\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCytoplasmic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eFer4_NifH\u003c/b\u003e\u0026nbsp;\u003cb\u003e(PF00142)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eABG74605.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27995.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e31.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e97.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCytoplasmic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eFer4_NifH\u003c/b\u003e\u0026nbsp;\u003cb\u003e(PF00142)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eADZ23568.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e232\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24942.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e34.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e99.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.079\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCytoplasmic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eFer4_NifH\u003c/b\u003e\u0026nbsp;\u003cb\u003e(PF00142)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eADZ23553.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25156.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e33.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e100.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.075\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCytoplasmic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eFer4_NifH\u0026nbsp;(PF00142)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eADZ23560.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25188.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e34.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e34.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e98.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.085\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCytoplasmic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eFer4_NifH\u0026nbsp;(PF00142)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eADZ23556.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e229\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24679.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e35.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e95.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.121\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCytoplasmic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eFer4_NifH\u0026nbsp;(PF00142)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eADZ23546.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e232\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24960.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e34.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e97.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.087\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCytoplasmic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eFer4_NifH\u0026nbsp;(PF00142)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eADZ23545.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e235\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25287.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e33.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e99.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.067\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCytoplasmic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eFer4_NifH\u0026nbsp;(PF00142)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSCN47971.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25156.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e32.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e100.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.077\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCytoplasmic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eFer4_NifH\u0026nbsp;(PF00142)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eADZ23543.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e229\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24665.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e34.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e95.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.121\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCytoplasmic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eFer4_NifH\u0026nbsp;(PF00142)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eADZ23542.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e233\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25073.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e34.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e99.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.070\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCytoplasmic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eFer4_NifH\u0026nbsp;(PF00142)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eADZ23544.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25188.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e34.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e98.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.085\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCytoplasmic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eFer4_NifH\u0026nbsp;(PF00142)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eADZ23535.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e233\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25057.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e34.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e99.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.094\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCytoplasmic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eFer4_NifH\u0026nbsp;(PF00142)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eACO90391.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e246\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26233.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e31.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e98.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.096\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCytoplasmic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eFer4_NifH\u0026nbsp;(PF00142)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAAK53549.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e245\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26249.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e32.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e98.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.099\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCytoplasmic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eFer4_NifH\u0026nbsp;(PF00142)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAHL17213.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e239\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25656.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e32.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e102.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCytoplasmic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eFer4_NifH\u0026nbsp;(PF00142)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAHL17212.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e245\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26513.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e35.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e96.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.112\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCytoplasmic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eFer4_NifH\u0026nbsp;(PF00142)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eADI72792.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e236\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25401.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e33.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e100.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.050\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCytoplasmic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eFer4_NifH\u0026nbsp;(PF00142)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eABG74605.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27995.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e31.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e97.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e-0.110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCytoplasmic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCytoplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003eFer4_NifH\u0026nbsp;(PF00142)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe molecular weights of these proteins exhibit a range from 24.66 to 31.99 KDa, with amino acid residue lengths spanning from 229 (ADZ23556.1) to 297 (AUX79023.1). Notably, all nitrogenases demonstrate acidic, as reflected in the theoretical pI values primarily falling between 4.18 and 4.8. Furthermore, the proteins exhibit high stability, indicated by instability indices consistently below 40. The aliphatic index, varying from 95.85 to 102.05 across all chosen sequences, suggests notable thermostability.\u003c/p\u003e \u003cp\u003eThe extinction coefficient, consistently light absorption at 280 nm in water, reveals a reduction in all Cys residues. Furthermore, the GRAVY values for all nitrogenase proteins range from \u0026minus;\u0026thinsp;0.050 to -0.129, suggesting a favorable interaction with water and classifying them as hydrophilic.\u003c/p\u003e \u003cp\u003eA conserved motif search highlights that all proteins possess a domain belonging to the Fe4_NifH family (4Fe-4S iron-sulfur cluster proteins: PF00142), as illustrated in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Predictions of our protein sequence\u0026rsquo;s subcellular localization by PSLpred indicate a presence in the cytoplasmic matrix with an accuracy of 98.1%. Cello further corroborates this, predicting the protein sequence to be cytoplasmic with a reliability score of 4.711 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The analysis of amino acids composition in the query sequence (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) underscores the dominance of four residues: alanine (A), glycine (G), and glutamic acid (E), with 10.2, 9.8, 8.9, and 8.5%, respectively.\u003c/p\u003e\u003cdiv\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003e3.3. Secondary Structure\u003c/h2\u003e\n \u003cp\u003eSecondary structures of all nitrogenase sequences were predicted using the SOPAM tool, and percentages of alpha-helix, extended strand, beta-turn, and random coils are provided in Table\u0026nbsp;3. Among the 22 nitrogenase genes, alpha helices predominated in 16 strains, with the highest value recorded in ACO90391.1 at 42.26%. Additionally, random coils were predominant in 6 strains, ranging from 31 (P19068.2) to 35.62 (ADZ23535.1). Our query sequence (QSZ40499.1) demonstrated 34.89% amino acids in random coils, 34.47% in alpha-helices, 19.57% in extended sheets, and 11.06% in the beta-turn region (Table\u0026nbsp;3). Further insight into the query sequence's secondary structure and its graphical representation was obtained through PSIPRED (Figure S2), and this secondary analysis was extended to the remaining sequences (Figure S1).\u003c/p\u003e\n \u003cdiv\u003e\n \u003cp\u003eTable 2 Composition of amino acid of query sequence\u0026nbsp;\u003c/p\u003e\n \u003cdiv align=\"left\"\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/div\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 3\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003ePredicted secondary structure content of 22 nitrogenase proteins (SOPMA)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNo.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAcces\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAlpha helix\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eExtended stand\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBeta turn\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRandom coil\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSZ40499.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.89\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP19068.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAUX79023.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.66\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eACE82208.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABG74605.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADZ23568.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADZ23553.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADZ23560.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADZ23556.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36.68\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADZ23546.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.62\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADZ23545.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSCN47971.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.48\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADZ23543.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADZ23542.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADZ23544.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADZ23535.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.62\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eACO90391.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAAK53549.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAHL17213.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAHL17212.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eADI72792.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABG74605.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003e3.4. 3D Structure Prediction Using Homology Modeling\u003c/h2\u003e\n \u003cp\u003eProtein homology modeling and evaluation were systematically conducted for all five typical proteins of \u003cem\u003eEnsifer fredii\u003c/em\u003e using the BLASTp tool [31]. Templates were chosen by identifying sequences similar to the query (QSZ40499), and phyre2, RaptorX, and Trosetta suggested the most appropriately matched template 3D protein models. Intriguingly, the AlphaFold and AlphaFold 2 server generated a model without any homolog or reference protein.\u003c/p\u003e\n \u003cp\u003eIn this comprehensive evaluation, four models chosen for this study (ABG74605, ACE82208, AUX79023, and sp|P19068) from the Alphafold server, exhibiting a high degree of similarity to our sequence (QSZ40499), displyed quality factor values ranging from 96.42–95.71%. Notably, our protein (QSZ40499) surpassed all, registering the highest percentage at 99.06%, thereby confirming its high-resolution structure.\u003c/p\u003e\n \u003cp\u003eAlphaFold consistently produces high-quality models with excellent ERRAT scores (95.71%-99.07%) and VERIFY 3D values (~ 88%-93%). In comparison, AlphaFold 2 also performs well, but VERIFY 3D scores drop slightly (~ 70%-78%), suggesting marginally reduced model quality.\u003c/p\u003e\n \u003cp\u003eBased on the Ramachandran Plot analysis, the AlphaFold-generated model exhibited the maximum number of residues in favored regions and the minimum number of residues in disallowed regions, making it more acceptable than the other models (Table\u0026nbsp;4).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 4\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003e3D Modeling score of predicted models of Nitrogenase enzyme using multiple server\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"11\"\u003e\u003c/colgroup\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eServer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" rowspan=\"2\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ePROVE (Outlier)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZ score\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eERRAT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVERIFY 3D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eRamashadran PLOT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eQuality Factor\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e3D-ID Score\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMost Favored regions\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAdditional allowed region (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenerally allowed region (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eDisallowed region (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\" rowspan=\"5\"\u003e\n \u003cp\u003eAlphaFold\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABG74605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3,2% (35)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,519\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96.4286\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e88.85%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,957\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,043\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eACE82208\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3,9% (37)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,538\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97.9839\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.66%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,942\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,058\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAUX79023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3,2% (35)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98.2394\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e86.15%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,946\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,054\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSZ40499\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3,7% (27)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,563\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.0698\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93.19%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,966\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,034\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esp|P19068\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3,2% (35)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,526\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95.7143\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e88.51%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,957\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,043\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\" rowspan=\"5\"\u003e\n \u003cp\u003eAlphaFold 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABG74605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98.8048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e78.54%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,924\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,076\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eACE82208\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98.4064\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e78.16%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,929\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,067\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAUX79023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97.5610\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e78.11%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,934\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,062\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSZ40499\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98.2143\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70.21%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,931\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,069\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esp|P19068\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97.5524\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e77.70%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,938\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,058\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\" rowspan=\"5\"\u003e\n \u003cp\u003ePhyre 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABG74605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e87.8136\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e88.89%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,876\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,116\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eACE82208\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e84.1897\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93.49%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,866\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,121\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAUX79023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e86.2454\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90.97%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,891\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,097\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSZ40499\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e91.9643\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.36%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,862\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,123\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esp|P19068\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e87.8136\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e88.89%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,876\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,116\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\" rowspan=\"5\"\u003e\n \u003cp\u003eRapptorX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABG74605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11,5% (102)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,877\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e85.7708\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90.80%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,906\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,085\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eACE82208\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9,9% (89)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,723\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92.8854\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92.72%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,911\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAUX79023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11,7% (103)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,791\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e88.5375\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94.25%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,888\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,085\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSZ40499\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92.3767\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e83.40%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,837\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,138\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esp|P19068\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11,1% (115)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,761\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90.9091\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90.20%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,864\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,113\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\" rowspan=\"5\"\u003e\n \u003cp\u003eTRosetta\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABG74605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5,3% (48)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,131\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90.9091\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95.79%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,929\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,067\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eACE82208\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6,4% (57)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,142\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94.0476\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96.17%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,929\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,062\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAUX79023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5,8% (60)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e81.9444\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e91.22%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,911\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,074\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,016\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSZ40499\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6,3% (45)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,172\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.2377\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95.74%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,936\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,054\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esp|P19068\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6% (62)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,078\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e85.6115\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e86.82%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,922\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,074\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe selected model (AlphaFold) showcased residues in favored regions ranging from 94.2% (ACE82208) to 96.6% (QSZ40499), with corresponding percentages in allowed regions ranging from 3.4% (QSZ40499) to 5.8% (ACE82208). No residues were identified in the disallowed regions across all sequences, with the overall quality factor of ERRAT values ranging between 95.6% and 99.06% (Table\u0026nbsp;4).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003e3.5. Refinement Structure\u003c/h2\u003e\n \u003cp\u003eAs a crucial step to refine the precision of initial structures and rectify local inaccuracies, the predicted 3D structure underwent scrutiny. The refined structure from the AlphaFold server was subject to quality assessments through ERRAT and Ramachandran plots. The revised structure's Ramachandran plot demonstrated an absence of residues in forbidden regions (Table\u0026nbsp;5, Figure S4, S6). Notably, the 3Drefine server emerged as the optimal choice for our query, evident in Z scores ranging from 0.057 to 0.277 and ERRAT (Overall Quality Factor) values between 98.8% and 100% (Table\u0026nbsp;5). This rigorous refinement process enhances the reliability of the structural insights gained from the study.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 5\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eRefinement score of predicted models of Nitrogenase enzyme using multiple server\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"10\"\u003e\u003c/colgroup\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eServer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePROVE (Outlier)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZ score\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eERRAT (Overall Quality Factor)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eRamashadran PLOT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQuality Factor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3D-ID Score\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMost Favored regions (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdditional allowed region (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGenerally allowed region (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDisallowed region (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003e3Drefine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABG74605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,057\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e88.85%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94,6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5,4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eACE82208\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,037\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,277\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98,8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97.32%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93,3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6,7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAUX79023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3,6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98,95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.86%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93,8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6,2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSZ40499\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,224\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99,55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96.17%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95,6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4,4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esp|P19068\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2,6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,251\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99,29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e88.85%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95,3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4,7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003eDeeprefiner\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABG74605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,028\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,442\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.53%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93,8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6,2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eACE82208\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,032\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97,16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98.47%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e91,1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8,9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAUX79023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2,50%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,416\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96,099\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92.91%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e91,4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8,6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSZ40499\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3,1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e85.53%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e91,6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8,4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esp|P19068\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3,4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98,58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.19%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94,6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5,4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003eGalaxy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABG74605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98,93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e88.18%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96,9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3,1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eACE82208\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,047\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98,78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92.72%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95,5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4,5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAUX79023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95,75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.86%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97,3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2,7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQSZ40499\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,579\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98,61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90.21%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97,5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2,5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esp|P19068\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95,39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.19%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97,7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2,3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003e3.6 Molecular Dynamics Simulation\u003c/h2\u003e\n \u003cp\u003e\u003cstrong\u003eStructural Stability and Flexibility\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe molecular dynamics (MD) simulations provided valuable insights into the structural stability of the nitrogenase Fe protein from \u003cem\u003eEnsifer fredii\u003c/em\u003e. The Solvent Accessible Surface Area (SASA) (Fig.\u0026nbsp;2A) exhibited moderate fluctuations throughout the simulation, reflecting the protein’s conformational flexibility while maintaining overall structural integrity. The trend in SASA suggests that the protein stayed within a defined range of solvent exposure, indicating reasonable stability.\u003c/p\u003e\n \u003cp\u003eThe temperature profile (Fig.\u0026nbsp;2B) remained stable, with only minor fluctuations, suggesting that the system was well-equilibrated. Additionally, the root mean square deviation (RMSD) showed that the protein conformation reached a stable plateau after an initial rise, confirming that the system achieved equilibration. Minor fluctuations around the plateau reflect routine conformational adjustments without compromising structural integrity.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eProtein Flexibility and Conformational Dynamics\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe root mean square fluctuation (RMSF) analysis (Fig.\u0026nbsp;2D) revealed regions of the protein with higher flexibility. These regions may correspond to active sites or other functionally relevant areas of the protein that undergo conformational changes during nitrogenase activity. When RMSF was compared with SASA data, regions displaying high flexibility also demonstrated increased solvent exposure (Fig.\u0026nbsp;2C), indicating that flexible areas of the protein may be more accessible for functional interactions.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCompactness and Thermodynamic Stability\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe radius of gyration (Rg) plot (Fig.\u0026nbsp;2G) showed that the overall compactness of the protein was maintained, with the Rg remaining relatively constant throughout the simulation. Although some anisotropy was observed in the dimensions Rgx, Rgy, and Rgz, these fluctuations were minor and did not significantly impact the structural integrity. The potential energy profile also plateaued after a period of equilibration, confirming that the protein achieved a stable energy state (Figs.\u0026nbsp;2E and 2F). The overall energy conservation during the MD simulation was reflected in the Gromacs energy plot (Fig.\u0026nbsp;2H), suggesting the system remained thermodynamically stable.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eNitrogenase serves a crucial role in converting atmospheric nitrogen dioxide to ammonia (NH3). However, the Mo-Fe-S complex nitrogenase enzyme is permanently inactivated by O2.\u003c/p\u003e \u003cp\u003eTo counter this, free-living diazotrophs employ respiratory protection to lower internal oxygen concentration, while plant polysaccharides in bacteroids maintain rapid aerobic respiratory turnover to support nitrogenase activity, potentially producing reactive oxygen species (ROS) as byproducts. Notably, three distinct nitrogen-fixing systems\u0026mdash;Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase\u0026mdash;have been identified, each composed of a two-component complex metalloenzyme system with dinitrogenase reductase as an iron protein and dinitrogenase as a metal cofactor [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the quest for a deeper understanding of nitrogenase, \u003cem\u003ein silico\u003c/em\u003e protein modeling has emerged as a cost-effective and expeditious alternative to experimental approaches [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Computational tools now play an increasingly pivotal role in navigating sequence space and enhancing laboratory evolution efficiency. Additionally, Sefdi et al. asserted the efficacy of bioinformatics tools as an effective bridge connecting protein sequences to their 3D structures.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Physiochemical Characterization\u003c/h2\u003e \u003cp\u003eA computationally based investigation into the physicochemical behavior of nitrogenases from several \u003cem\u003eEnsifer\u003c/em\u003e spp. was performed, and this analysis provided a theoretical insight into protein nature. The pH at which a protein attains neutrality, referred to as the isoelectric or isoionic point [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], was determined across 22 strains, yielding scores ranging from 4.84 to 5.18. This range suggests a moderate acidity in naturally occurring nitrogenases. The stability of proteins, measured by the instability index (II), designates values exceeding 40 as indicative of instability [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Interestingly, all nitrogenase strains of E. \u003cem\u003efredii\u003c/em\u003e exhibited instability scores below 40, signifying the stability of this protein.\u003c/p\u003e \u003cp\u003eAccording to IKAI (1980), the aliphatic index (AI), influencing a protein's thermal stability, gauges the proportion of a protein's relative volume occupied by aliphatic amino acids in the side chain. Demonstrating exceptional thermostability, all enzymes exhibited an AI value exceeding 95.85%. \u003cem\u003eEnsifer fredii\u003c/em\u003e, among the limited bacteria displaying such characteristics, showcased the highest concentrations of alanine (10.2%), glycine (9.8%), and leucine (8.9%). This composition likely contributed to the heightened thermostability observed in \u003cem\u003eE. fredii\u003c/em\u003e. The enzymes were confirmed as thermostable, characterized by elevated aliphatic index values and a substantial percentage of alpha-helix structures.\u003c/p\u003e \u003cp\u003eThe interplay between water and protein was elucidated through the GRAVY metric. A positive GRAVY value denotes hydrophobicity, while a negative value signifies enhanced hydrophilic interactions with water. In addition, the polar characteristics of the hydrophilic amino acids glycine, glutamic acid, lysine, aspartic acid, and serine make them more likely to interact in aqueous environments. Cystidine residues are crucial in creating disulfide connections between different protein components. Disulfide bonds are crucial for protein folding and stabilization of the unfolded state by reducing entropy. However, in most cases, disulfide bridges are lacking due to cysteine residues' extremely low occurrence [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Secondary Structural Analysis\u003c/h2\u003e \u003cp\u003eThe prediction of secondary protein structures from sequences serves as a crucial link connecting primary and tertiary structure predictions [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Random coil (34.89%) and alpha helix (34.47%) were the two most common secondary structures, while extended stand (19.57%) and beta-turn (11.06%) were the other configurations [Figure S3]. High scores in random coils signify the absence of regular secondary structures, providing flexibility for conformational changes, such as enzyme turnover [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Meanwhile, extended strands and alpha-helices contribute to protein stability. In a parallel investigation by Satyanarayana et al. Nif-A sequences in \u003cem\u003eBradyrhizobium japonicum\u003c/em\u003e, \u003cem\u003eRhizobium leguminosarum\u003c/em\u003e, and \u003cem\u003eMisorhizobium ciceri\u003c/em\u003e showcased dominance in random coil, extended strand, and beta-twist configurations during secondary structure analysis. This reinforcing the consistency of our findings [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe oxygen sensitivity of NifH proteins appears to correlate with the presence of alanine and glycine residues, indicating a potantiel connection to the coordination of metal ions and the redox status of the proteins [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Employing \u003cem\u003ein silico\u003c/em\u003e protein structure research emerges as a valuable method for exploring the structural and functional characteristics of proteins [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAcquiring twenty-two sequences (both amino acids and their corresponding gene sequences) from NCBI, our phylogenetic analysis of cDNA for nitrogenase proteins revealed a congruent clustering with the protein comparison (Figure S3). The phylogenetic tree, indicating two distinct groups within the selected strains. This result aligns with the approaches of other researchers [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], who used a similar method to assess associations between taxa protein sequences and their corresponding cDNA sequences. Notably, the high reliability of the tree is underscored by bootstrap values ranging from 98\u0026ndash;100%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Tertiary Structure Analysis\u003c/h2\u003e \u003cp\u003eConfirming information derived from NMR/X-ray crystallography-based approaches poses a significant challenge in predicting a protein's three-dimensional (3D) model through \u003cem\u003ein silico\u003c/em\u003e analysis [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Due to the absence of experimental data in the protein data bank, tertiary structure prediction homology modeling was employed to predict the 3D structure of the nitrogenase protein. RaptorX, TrRosetta, and Phyre2 created a structural model by choosing the optimal template for modeling, whereas AlphaFold created a protein model based solely on the amino acid structure without a template or homologous structure.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEnsifer fredii\u003c/em\u003e was chosen as a representative species for homology protein modeling and submission, clarifying the nitrogenase protein structure of \u003cem\u003eEnsifer\u003c/em\u003e spp. The selection was based on Z score, an overall quality factor from the SAVES server, and the Ramachandran plot. Homology modeling was conducted for all five proteins (see figures S3 and S5, Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Phyre2's alignment file was adjusted to accommodate the overlap between the secondary structure elements of the template and the predicted secondary structure profile of the sequence. On the other hand, TrRosetta and RaptorX utilized random generation and spatial resistance to produce five initial protein models.\u003c/p\u003e \u003cp\u003eThe quality of the model was assessed using the Ramachandran plot, where more than 90% of residues were located in the preferred regions, indicating high quality [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Examining the PDB structure of \u003cem\u003eEnsifer fredii\u003c/em\u003e (QSZ40499) protein, the Ramachandran plot revealed that 97.9% of residues occupied the most preferred region. A high-quality model was further indicated by a Z-score falling within the range of 0\u0026ndash;1 [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] and 1 [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] compared to a non-redundant PDB structure set.\u003c/p\u003e \u003cp\u003eComputational methods were employed to examine and analyze the tertiary structures of the selected nitrogenase enzymes. Quality parameters including ERRAT, Z-score, and the Ramachandran plot were utilized. A high-resolution structure, with an overall quality factor of 95% or higher, was considered good [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The retrieved Z-score mean and standard deviation from the SAVES server were 0.524 and 0.538, respectively, aligning closely with the expectation for a high-resolution structure (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. three homology modeling programs (Physical2, Trosetta, and RaptorX) were employed, along with the template-free approach of AlphaFold and AlphaFold 2. The results consistently affirmed the reliability and precision of the predicted structure by AlphaFold.\u003c/p\u003e \u003cp\u003eAlthough our model includes conserved nucleotide-binding motifs, the absence of explicit ATP/ADP coordinates in the AlphaFold structure may limit direct visualization of the nucleotide-binding conformation. Future studies incorporating ligand-docking simulations could address this gap.\u003c/p\u003e \u003cp\u003eAlthough our model includes several critical residues for nucleotide binding, it lacks the critical N-terminal sequence stretch required for this binding. We believe that this constraint will not jeopardize the current investigation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.4. Model Refinement\u003c/h2\u003e \u003cp\u003eRefining the projected 3D protein model is crucial to enhance the prediction models' quality. Three servers\u0026mdash;3Drefiner, Galaxy, and Deeprefiner\u0026mdash;were employed for this purpose, aiming to advance the experimental accuracy of models for subsequent computational investigations [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. After analyzing the Ramachandran plot for the PDB structure of selected queries. The Galaxy server was chosen due to the high percentage range (95,5\u0026ndash;97,7%) of residues present in the most favored region. The 3Drefine server appears to be the best server of refinement to our query based on a Z score between (0,057-0.277) and ERRAT (Overall Quality Factor) between (98.8\u0026ndash;100) (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Figure S4). The increased overall model quality factor obtained through ERRAT analysis for our query (QSZ40499) indicated structural improvement, rendering it suitable for additional docking studies (presence of more than 90% residues in the favored (red) region of the Ramachandran plot [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The model's accession number is PM0084218, and it was uploaded to the Protein Model Database (PMDB) database in pdb format after being refined and reviewed (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinformatics.cineca.it/PMDB/index.php\u003c/span\u003e\u003cspan address=\"https://bioinformatics.cineca.it/PMDB/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eProtein interactions with ligands, ions, and other proteins play a crucial role in determining protein activity. However, the focus on protein structure is fundamental as the initial step for computational functional investigations [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. For an improved 3D model, considerations of oligomeric states and protein-ligand complexes are essential, reflecting real-world protein interactions with ligands such as ions, inhibitors, and peptides. Neglecting these intricate molecular systems could result in the refinement of artificial protein models.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Molecular Dynamics Simulation\u003c/h2\u003e \u003cp\u003eThe MD simulation results reveal that the nitrogenase Fe protein exhibits a delicate balance between structural stability and functional flexibility. The moderate SASA fluctuations suggest that while the protein remains solvent-exposed in certain regions, its core structure remains robust. This solvent exposure, particularly in regions with higher RMSF values, may be critical for the protein's biological function, enabling the necessary conformational changes required during nitrogen fixation [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe RMSD data further support the notion of a stable conformation after the initial equilibration phase, with only minor fluctuations indicative of routine conformational sampling [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The plateauing of the RMSD and potential energy highlights that the system has reached a stable energetic and structural state [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The flexibility observed in the RMSF data corresponds to regions that might be involved in dynamic interactions, such as substrate binding or catalysis [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. These flexible, solvent-exposed regions could play a critical role in the protein's functional mechanisms. The combination of high flexibility and solvent exposure often corresponds to dynamic functional regions, which are essential for the enzymatic activity of nitrogenase. The constant radius of gyration suggests that the overall compactness of the protein is preserved, indicating that the core structure remains stable. This compactness, despite minor anisotropic variations, aligns with the energy conservation observed throughout the simulation, underscoring the protein\u0026rsquo;s ability to remain structurally intact while maintaining flexibility where needed.\u003c/p\u003e \u003cp\u003eThe molecular dynamics simulations reveal that the [Fe-S] cluster plays a pivotal role in stabilizing the nitrogenase Fe protein\u0026rsquo;s core, as indicated by RMSF values below 1.5 \u0026Aring; in the cluster region (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). This stability, driven by the cluster\u0026rsquo;s coordination, likely supports its function in electron transfer during nitrogen fixation, particularly under redox stress. Meanwhile, the flexibility of solvent-exposed loops (RMSF\u0026thinsp;\u0026gt;\u0026thinsp;3 \u0026Aring;) suggests these regions contribute to the protein\u0026rsquo;s adaptability, potentially aiding interactions critical for activity. This interplay between a rigid core and dynamic periphery underscores the structural balance essential for nitrogenase function.\u003c/p\u003e \u003cp\u003eThe MD simulations demonstrate that the nitrogenase Fe protein from \u003cem\u003eEnsifer fredii\u003c/em\u003e can maintain a stable structure with the necessary flexibility for functional activity. These properties likely contribute to its role in nitrogen fixation, where dynamic interactions and conformational adjustments are crucial for optimal performance. Future experimental studies may focus on further exploring these flexible regions to better understand their role in nitrogenase function.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eThis study provides significant in silico insights into the nitrogenase enzyme of \u003cem\u003eE. fredii\u003c/em\u003e, emphasizing its physicochemical and structural properties. Key findings include sequence lengths ranging from 229\u0026ndash;297 amino acids across the analyzed NifH proteins, with our primary focus on QSZ40499 (residues 32\u0026ndash;266), demonstrating a predominance of hydrophilic residues (34.89% random coils) and dynamic structural behavior essential. AlphaFold modeling, validated by Z-scores and ERRAT metrics, demonstrated the enzyme\u0026rsquo;s balance between stability and flexibility, essential for nitrogen fixation. Molecular dynamics simulations revealed solvent-exposed flexible regions facilitating functional activity. These findings establish a foundation for future experimental studies and biotechnological applications aimed at enhancing nitrogen fixation for agricultural and industrial purposes.\u003c/p\u003e \u003cp\u003eFuture studies should prioritize experimental validation of the nitrogenase enzyme\u0026rsquo;s catalytic properties. Additionally, integrating structural insights with biotechnological strategies can optimize nitrogen fixation for sustainable agriculture and industry.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe conceptualization of the study was proposed by Mohamed Hnini and Karim Rabeh, both of whom made equal contributions to result analysis and manuscript writing. All authors have reviewed and approved the final manuscript.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eThere is no funding to report for this study.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article as tables or figures.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/h4\u003e\n\u003ch4\u003eThe authors declare no competing interests.\u003c/h4\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEthical Approvals\u003c/strong\u003e\u003c/p\u003e\n\u003ch4\u003eNot applicable.\u003c/h4\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eInformed Consent\u003c/strong\u003e\u003c/p\u003e\n\u003ch4\u003eNot applicable.\u003c/h4\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBurris, R.H.; Roberts, G.P. 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Sel.\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e35\u003c/em\u003e, gzac001, doi:10.1093/protein/gzac001.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Chemistry](https://link.springer.com/journal/44371)","snPcode":"44371","submissionUrl":"https://submission.nature.com/new-submission/44371/3","title":"Discover Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Nitrogenase, Ensifer fredii, Modeling, Alphafold, In silico, MD-simulation","lastPublishedDoi":"10.21203/rs.3.rs-5209284/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5209284/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe nitrogenase Fe protein, central to nitrogen fixation, has been extensively studied, However, the X-ray structure of the Nase-Fe protein from \u003cem\u003eEnsifer fredii\u003c/em\u003e is essential for understanding its function and stability, yet this structure is absent from the database. This study aimed to analyze the structural features of the NifH protein (EC 1.18.6.1, nitrogenase component II)) from \u003cem\u003eEnsifer fredii\u003c/em\u003e using an \u003cem\u003ein silico\u003c/em\u003e approach. Utilizing diverse biocomputational tools, we examined the primary, secondary, and tertiary structures of 22 NifH proteins, focusing on QSZ40499. Results indicate that QSZ40499 is a highly stable, hydrophilic, cytoplasmic protein with a molecular weight of 25.34 kDa. Its functional motif corresponds to the Fe4 family domain, and its secondary structure comprises 34.89% random coils and 34.47% alpha helices. 3D modeling via AlphaFold was validated using structural quality assessment servers and refined using the 3DRefiner server, supported by Z-scores and ERRAT Factor metrics. Molecular dynamics simulations revealed that QSZ40499 maintains structural stability and functional flexibility, essential for dynamic interactions during nitrogen fixation. This work provides valuable insights into the structural attributes of nitrogenase enzymes, advancing bioinformatics research and understanding of nitrogen fixation mechanisms.\u003c/p\u003e","manuscriptTitle":"In Silico Exploration of Nitrogenase in Ensifer fredii Unraveling Structural Homology Functional and Phylogenetic Relationships and Molecular Dynamics ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-01 13:56:17","doi":"10.21203/rs.3.rs-5209284/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-16T06:54:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-09T17:38:01+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-01T11:22:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-01T07:03:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"85755724237798369609367413833979284916","date":"2025-04-01T06:43:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296856059040742342782848840920628977561","date":"2025-04-01T04:40:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-31T19:00:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-26T06:41:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Chemistry","date":"2025-03-22T23:43:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Chemistry](https://link.springer.com/journal/44371)","snPcode":"44371","submissionUrl":"https://submission.nature.com/new-submission/44371/3","title":"Discover Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"470799a7-18f6-462c-94e3-6bc91d700367","owner":[],"postedDate":"April 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-05-12T03:53:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-01 13:56:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5209284","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5209284","identity":"rs-5209284","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}