In situ architecture of the nuclear pore complex of the higher plant Arabidopsis thaliana

In situ architecture of the nuclear pore complex of the higher plant Arabidopsis thaliana | 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 Article In situ architecture of the nuclear pore complex of the higher plant Arabidopsis thaliana Hugo Germain, Ingrid Berenice Sanchez Carrillo, Patrick Hoffmann, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6197803/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Oct, 2025 Read the published version in Nature Plants → Version 1 posted You are reading this latest preprint version Abstract The nucleus is delimited by the nuclear envelope (NE), where nuclear pore complexes (NPCs) are embedded. While this complex has been well studied in vertebrates, yeast, and, more recently, in algae, in situ structural data of higher plants is still missing. Here, we show that many individual nucleoporins of Arabidopsis thaliana and human present high structural similarity. We report a first higher plant in situ NPC structure, derived from A. thaliana root protoplasts using cryo electron tomography, subtomogram averaging and homology-based integrative modeling. We present a plant NPC model based on predicted models of A. thaliana NUPs identified by mass spectrometry. The plant NPC scaffold exhibits differences in diameter and height to C. reinhardtii, while sharing some structural features with H. sapiens NPCs. Notably, we observed that the A. thaliana NPC contains NUP155 connectors like the H. sapiens NPC, which may explain the height difference when compared to the C. reinhardtii NPC. Biological sciences/Cell biology/Cell signalling/Nuclear receptors Biological sciences/Plant sciences/Plant cell biology Higher plants subtomogram averaging cryo-ET cryo-FIB nucleoporin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Nuclear pore complexes (NPCs) are large multi-protein complexes involved in the selective import and export of macromolecules passing through the nuclear envelope (NE) 1 . Exclusive to eukaryotic organisms, NPCs play crucial roles in regulating gene expression 2 , chromatin organization 3 , DNA repair, and RNA processing and quality control 4 , 5 . NPCs are organized into different subcomplexes 6 made of multiple copies (~ 1000 protein subunits in humans 7 ) of approximately 30 different proteins known as nucleoporins (NUPs) 8 . With a few exceptions, NUPs are largely conserved among eukaryotes 7 . Much of our understanding of the NPCs comes from various studies using electron microscopy (EM) 9 , which has continued developing and refining, allowing the study of a broad range of model species 10 – 16 . Cryo electron tomography (cryo-ET) studies have revealed that the overall architecture of the NPC forms a three-layered scaffold structure 7 , 14 , 15 , 17 , 18 , with an octagonal symmetry around the central transport channel 19 . These three layers consist of the three main rings: the nuclear ring (NR), situated on the nucleoplasmic side; the inner ring (IR), embedded at the fusion point of the inner and outer nuclear membrane 7 ; and the cytoplasmic ring (CR). Structural data for the NPC of model organisms such as yeast 10 , 20 , 21 , Chlamydomonas 13 , Xenopus 22 – 24 and human 25 are now available, showcasing a range of structural differences within their NPC scaffolds. However, technical hurdles have limited the use of cryo-ET for higher plants and our understanding of plant NPCs largely relied on mass spectrometry 12 and nucleoporin homology to better-characterized organisms. Recently we optimized the methodology to use cryo-ET to investigate the structural characteristics of protein complexes in Arabidopsis thaliana 26 , which we use herein to provide insights into the evolution and origin of the plant NPC in eukaryotic organisms. A. thaliana is a small rosette plant that was first adopted as a model organism for its utility in genetic studies. It features a quick generation time, a small size (minimizing the need for extensive growing facilities), ease of genetic transformation, and prolific seed production through self-pollination. All of which make it a great model in plant biology 27 , 28 . In contrast to many organisms, A. thaliana can withstand a high level of homozygosity and has a relatively small genome (132 Mbp) 29 , 30 . Despite its short life cycle, producing transgenic plants still requires several months 31 . To circumvent these delays, electroporation and polyethylene glycol (PEG)-based transfection of protoplasts have grown in popularity as a tool for transient expression of genetic material. Since then, protoplasts have been extensively used to study various aspects of plant physiology, cell ultrastructure, and genetics 32 . Protoplasts are isolated cells without cell walls, and the enzymatic removal of the cell wall, known as protoplasting, does not obscure cell type differences or prevent comparisons with whole tissues, as it preserves physiological responses and cellular activities 33 – 35 . While procedures have been developed for employing protoplasts for various purposes, the use of in situ cryo-ET to plant protoplasts was still limited until recently. In this study, we used a workflow using A. thaliana root protoplasts, cryo focused ion beam (cryo-FIB) milling 36 of vitrified protoplasts for cryo-ET, and combined it with subtomogram averaging (STA) to examine and reveal the first NPC structure from a higher plant within its cellular environment. We constructed a model for the A. thaliana NPC (AtNPC) based on the structure prediction of A. thaliana nucleoporin homologs using integrative modeling. The identified scaffold arrangement is comparable to the previously published NPC of the unicellular algae C. reinhardtii (CrNPC) 13 , however with an overall smaller diameter than the H. sapiens NPC (HsNPC) while maintaining a similar height and NUP155 connectors in the cytoplasmic ring. Results Mass spectrometry reveals thirty of the thirty-four NUPs of the plant NPC To identify the NUPs that make up the A. thaliana NPC we performed nuclear extraction 37 from root protoplasts followed with high-performance liquid chromatography and tandem mass spectrometry. Our results allowed us to identify thirty out of the thirty-four described A. thaliana nucleoporins 38 – 40 as being present in the root protoplasts (Table 1 ) which is commendable considering that established protocols detect around 25 NUP 39 . Among the identified NUPs, we detected all NUPs of the inner ring, and most NUPs of the Y-complex. Only, NUP50a, NUP136, CG1, and NUP98b could not be detected with a false discovery rate (FDR) of 1%. NUP50a and NUP136 are usually found at the nuclear basket, while CG1 is located on the cytoplasmic side. NUP98b is a peripheral NUP located on both the cytoplasmic and nuclear sides of the central channel 3 . A plausible explanation for the undetected NUPs, is that they may have become dissociated from the nuclear pore complex during the process of nuclear extraction and sample preparation. Our workflow demonstrated that we were able to detect most NUPs of the nuclear basket, nuclear and peripheral sides and use this information for further understanding of the elements that conform the plant NPC. Table 1 Mass spectrometry identification of A. thaliana NUPs from nuclear extracts purified from A. thaliana root protoplasts. List of A. thaliana NUPs identified using mass spectrometry. The last column explains whether the proteins were detected or partially detected in the three technical replicates. Accession numbers were obtained from the Uniprot database ( https://www.uniprot.org/ ). empAI values were determined by carrying out a Mascot search (Matrix Science) and analysis with the Scaffold software (version 5.2.2, Proteomes Software Inc., Portland, USA). Protein Accession Number emPAI value Sample 1 emPAI value Sample 2 emPAI value Sample 3 Std deviation Sample 1 Std deviation Sample 2 Std deviation Sample 3 Presence ALADIN AAAS_ARATH 0.4000 0.3560 0.1987 0.1267 0.1746 0.0782 3/3 NUP50C Q93ZH3_ARATH 0.0914 0.0000 0.0000 0.0033 0.0000 0.0000 1/3 HOS1 HOS1_ARATH 0.5453 0.7713 0.6147 0.0792 0.0665 0.0990 3/3 NUP205 A0A1P8BGZ1_ARATH 0.5220 0.5757 0.6167 0.1031 0.1561 0.1902 3/3 GP210 GP210_ARATH 0.9063 1.3200 1.0507 0.3410 0.0300 0.1028 3/3 NUP35 NUP35_ARATH 0.1230 0.1827 0.0000 0.0046 0.0809 0.0000 2/3 NUP43 NUP43_ARATH 0.1127 0.4867 0.2793 0.1115 0.5187 0.0588 3/3 NUP50B NU50B_ARATH 0.0603 0.0000 0.0000 0.1045 0.0000 0.0000 1/3 NUP54 NUP54_ARATH 0.1773 0.5193 0.0000 0.0601 0.1638 0.0000 2/3 NUP58 NUP58_ARATH 0.1572 0.0867 0.0182 0.0782 0.0891 0.0315 3/3 NUP62 NUP62_ARATH 0.3720 0.6040 0.0821 0.1005 0.1459 0.0037 3/3 NUP85 NUP85_ARATH 0.6697 1.0183 0.6583 0.1230 0.2886 0.2657 3/3 NUP88 NUP88_ARATH 0.1689 0.1087 0.1398 0.0645 0.0586 0.0632 3/3 NUP93A NP93A_ARATH 0.7823 0.6067 0.3477 0.0926 0.3361 0.1736 3/3 NUP93B NU93B_ARATH 0.1094 0.0654 0.0948 0.0777 0.0277 0.0839 3/3 NUP96 NUP96_ARATH 0.1643 0.3200 0.1400 0.0609 0.1416 0.0320 3/3 NUP98A NU98A_ARATH 0.0542 0.0764 0.0189 0.0235 0.0568 0.0164 3/3 NUP107 NU107_ARATH 0.1710 0.2847 0.2340 0.0879 0.0081 0.0187 3/3 NUP133 NU133_ARATH 0.2017 0.3237 0.2163 0.0736 0.1061 0.0097 3/3 NUP155 NU155_ARATH 0.6537 0.7207 0.4023 0.0672 0.1554 0.0668 3/3 NUP160 NU160_ARATH 0.2690 0.2323 0.3240 0.0501 0.1061 0.0901 3/3 NUP214 NP214_ARATH 0.0000 0.0080 0.0155 0.0000 0.0139 0.0160 2/3 NUA NUA_ARATH 0.4960 0.6093 0.4250 0.1025 0.1793 0.0267 3/3 NUP188 F4JUG3_ARATH 0.1155 0.1032 0.0177 0.0360 0.0560 0.0069 3/3 NDC1 Q8LAF4_ARATH 0.2993 0.1712 0.1066 0.0589 0.0878 0.0930 3/3 GLE1 GLE1_ARATH 0.0000 0.0000 0.0135 0.0000 0.0000 0.0233 1/3 RAE1 RAE1_ARATH 0.4657 1.0457 0.1330 0.0692 0.9646 0.0454 3/3 SEH1 SEH1_ARATH 0.4093 0.6237 0.2433 0.1678 0.2010 0.0663 3/3 SEC13A SC13A_ARATH 0.5030 0.1993 0.1254 0.1828 0.0907 0.0595 3/3 SEC13B SC13B_ARATH 1.7567 0.9040 0.6103 0.5021 0.2272 0.1463 3/3 Structural models of individual NUPs of the Y-complex are conserved between A. thaliana and H. sapiens Of all the constituents that make up the NPC, the Y-complex (also known as the NUP84 complex in yeast or the NUP107–Nup160 complex in vertebrates) is probably the best studied and structurally characterized NPC substructure 7 . Consisting of 6 to 10 NUPs depending on the organism 41 , this complex is a main constituent of the cytoplasmic and nuclear ring 7 . Previous studies across different organisms 13 , 22 , 42 – 49 revealed the conservation of proteins and the architecture among eukaryotic species 4 , 5 , 50 . Based on the information from our mass spectrometry data, we compared the three-dimensional (3D) structures of the NUPs that make up the A. thaliana Y-complex to visualize the structural similarities between the NUPs that make up their respective Y-complexes. To achieve this, we set side by side (Fig. 2 ) the AlphaFold predictions 51 , 52 of the NUPs from the Y-complex from A. thaliana and H. sapiens NUPs and measured the similarity between these NUPs (TM-score) 53 to assess their structural similarity. Comparing the 3D structures of proteins represents an opportunity to gain a deeper understanding of their evolutionary relationship 54 . Table 2 TM-score values of A. thaliana and H.sapiens NUPs from the Y-complex. TM-scores from A. thaliana and H.sapiens NUPs that are present in the Y-complex of both organisms using the TM-score tool from the Zhang group 53 . ID accessions were obtained from the AlphaFold database ( https://alphafold.ebi.ac.uk/ 51,52,55 ). Protein AlphaFold ID TM-score A.thaliana NUP160 AF-F4IGA5-F1-v4 0.58283 H. sapiens NUP160 AF-Q12769-F1-v4 A.thaliana NUP85 AF-Q8RXH2-F1-v4 0.70345 H. sapiens NUP85 AF-Q9BW27-F1-v4 A.thaliana NUP43 AF-Q24JJ9-F1-v4 0.79516 H. sapiens NUP43 AF-Q8NFH3-F1-v4 A.thaliana SEH1 AF-Q93VR9-F1-v4 0.86101 H. sapiens SEH1 AF-Q96EE3-F1-v4 A.thaliana NUP96 AF-Q8LLD0-F1-v4 0.90428 H. sapiens NUP96 AF-P52948-F1-v4 A.thaliana SEC13B AF-O64740-F1-v4 0.95452 H. sapiens SEC13 AF-P55735-F1-v4 A.thaliana SEC13A AF-Q9SRI1-F1-v4 0.95315 H. sapiens SEC13 AF-P55735-F1-v4 A.thaliana NUP107 AF-Q8L748-F1-v4 0.57271 H. sapiens NUP107 AF-P57740-F1-v4 A.thaliana NUP133 AF-F4IGA5-F1-v4 0.39414 H. sapiens NUP133 AF-Q8WUM0-F1-v4 Our results confirmed that some components of the Y-complex that are found in vertebrates are broadly conserved among eukaryotic organisms as previously suggested 56 . We found that the A. thaliana Y-complex shares 8 out of 10 NUPs (NUP160, NUP85, NUP43, SEH1, NUP96, SEC13, NUP107, and NUP133) with that of H. sapiens. The remaining two NUPs, NUP37 and ELYS, appear to be absent from the plant NPC as they have not been described from previous plant mass spectrometry experiments 38 , 40 , 57 . However, based on sequence analysis the functional homolog of ELYS in plants is suspected to be HOS1 38 . These findings suggest that, despite more than 500 million years of evolutionary separation between plant and animal cells, most of their Y-complex is conserved, with only a few species-specific nucleoporins. The in situ NPC structure of higher plant cells revealed in A. thaliana root protoplasts To study plant NPCs in their native state, we utilized a recently established workflow 26 employing isolated root protoplasts from transgenic A. thaliana plants expressing RAE1-GFP 38 for in situ cryo-ET. In brief, we vitrified the isolated protoplasts on cryo-EM grids and intact nuclei protoplasts expressing RAE1-GFP were then identified using a cryo Correlative Light and Electron Microscopy (cryo-CLEM). Protoplast lamellae were prepared using an Aquilos cryo-FIB microscope. We specifically targeted areas of the nuclear envelope for tilt-series acquisition in a cryo Transmission Electron Microscope (cryo-TEM), followed by tomogram reconstruction and subtomogram averaging (STA). These methods allowed us to structurally analyze the A. thaliana NPC in situ . We acquired 37 tomograms of A. thaliana nuclear envelopes which contained a total of 79 NPCs to examine the in situ architecture of the plant NPC (Fig. 3 a). Using a previously established subtomogram averaging approach 16 , 50 we obtained the cryo-ET map of the A. thaliana NPC from these 79 NPCs at a resolution of 35Å for the focused maps of individual rings (Fig. 3 b, c). To better understand the eight-fold symmetrical scaffold architecture, we segmented the cryo-ET map into subcomplexes. On the cytosolic side, the CR of the A. thaliana NPC consists of one ring with eight copies of the Y-complex. In addition, each asymmetric unit contained density for the NUP214 complex and the NUP205-NUP93 complex (Fig. 3 d). In contrast, the NR comprises two copies of the Y-complex complex per asymmetric unit and density for the NUP205-NUP93 complex, resulting in a total of 16 Y-complexes for the NR that form two concentric rings of inner and outer Y-complexes. Both CR and NR show prominent densities for NUP155 connectors linking them to the eight spokes of the IR (Fig. 3 d). Having established the overall makeup of the A. thaliana NPC, we constructed the first structural model of a higher plant NPC, based on our cryo-ET map, using predicted models 51 of 20 A. thaliana NUPs, which we also previously had confirmed by mass spectrometry to be present in root protoplasts (Fig. 3 e). A. thaliana Y-complex is comparable to C. reinhardtii , but connector elements resemble those of H. sapiens NPC. The individual subcomplexes comprising the CR, IR and NR of the NPCs are mostly conserved across different species 58 . Similarly, the A. thaliana NPC subcomplexes exhibit high structural similarity to previously published NPC building blocks 13 . To better illustrate the similarities of the A. thaliana NPC scaffold and to highlight potential differences from the NPC structures of other species, we compared our A. thaliana NPC (AtNPC) structure to the NPC of the single cell algae C. reinhardtii (CrNPC) (EMD-4355 13 ) and the human in situ NPC structure from HEK293 cells (HsNPC) (EMD-14321 14 ). Beyond the apparent similarities in the overall architecture, some striking contrasts are evident between the AtNPC, the CrNPC, and the HsNPC. In comparison to the HsNPC, which features an alternative Y-complex arrangement and two concentric rings for either the CR and NR, the AtNPC is generally shorter in height. Nevertheless, AtNPCs are taller than the CrNPC. The presence of the connectors elements (NUP155) between the outer rings and the IR spoke, which are not clearly resolved in the CrNPC 13 , could contribute to this overall taller height (Fig. 3 e). We also observe variations in the average diameter of the NPC (measured from membrane to membrane) across the three organisms. The AtNPC has the smallest average diameter measuring approximately 70 nm between the membranes, HsNPCs from HEK293 cells measure an average diameter of 92 nm in diameter and the CrNPCs have the largest average diameter with 102 nm (Fig. 4 ). These differences could reflect different amounts of membrane tension under the given conditions between the three experiments. However, if these variations in diameter measurements and spatial dynamics reflect the architecture, they are likely to impact the function of nucleocytosolic transport as well as the osmotic response, possibly emphasizing the importance of understanding these differences 16 , 59 , 60 . The head-to-tail contact in the AtNPC CR Y-complex and NR inner Y-complex is formed between NUP133 α-solenoid domain and HOS1, while the head-to-tail contact in the human NPC is formed between NUP133 β-propeller domain and NUP160 (Fig. 5 , Fig. S1). Contrary to the human NPC, one copy of HOS1 appears present in the CR of the AtNPC while two HOS1 copies are present in the NR (Fig. 6 a, b). Moreover, two copies of a large nucleoporin likely representing NUP205 are in the CR of the AtNPC while only one copy is in the NR (Fig. 6 c, d). The C-terminal domain of NUP93A/B is positioned differently in the AtNPC compared to the C-terminal domain of NUP93 in the human NPC. There is no density in the AtNPC at the positions corresponding to NUP93 in the human NPC. The AlphaFold2 model of the interaction between NUP93/NUP93A/B, NUP133, and NUP107 looks different in human and A. thaliana . NUP107 in A. thaliana has an additional domain (not present in human NUP107) that interacts with NUP93A/B in A. thaliana (Fig. 6 e). The C-terminal domain of NUP93A/B seems to interact with NUP107 and NUP133 in the CR in the position modeled by AlphaFold2 (the density corresponding to NUP93A/B next to NUP107/NUP133). However, NUP93A/B is not present in the corresponding positions in the NR. There is another region in the NR that could possibly be occupied by NUP93A/B, but this interaction is not modeled by AlphaFold (Supplementary Fig. 2). Discussion The presence of a double membrane layer separating the genetic material from the cytoplasm led to the formation of the nucleus, enabling nucleus-specific cellular processes. Direct passage through nuclear pores offers a great throughput, maintains semi-permeability and enables the controlling of molecular trafficking. The high degree of similarity among nuclear pores suggests they evolved from a common ancestor, later developing species-specific features. Despite the crucial importance of plants, technical limitations have hindered detailed investigation of their nuclear pore structure until recently. Here, we present the first detailed three-dimensional structure of the nuclear pore complex from a higher plant, providing insights into the evolutionary relationships that have shaped the NPC and its nucleoporins across and within kingdoms. Our mass spectrometry results enabled us to compare the NUPs present in both Arabidopsis NPC with published data from human NPC. We identified thirty out of the thirty-four described A. thaliana nucleoporins 22 , 38 – 40 as being present in the root protoplasts (Table 1 ) suggesting that our nuclei purification protocol and mass spectrometry protocol were equally or more efficient in comparison to previously published protocol aiming NUP identification 40 , 57 . The techniques used to isolate the nuclear fraction, such as centrifugation, mechanical disruption, and exposure to various buffers and reagents, might have disrupted the structural integrity of the NPC, leading to the detachment of the four missing nucleoporins. While these four NUPs have been detected in other published studies 38–40,61−65 , this difference could be due to our exclusive use of root protoplast cells, whereas other studies utilized whole A. thaliana plantlet, allowing for the detection of proteins present across multiple tissue or cell types. Specifically, for NUP136, it is known to be tethered to the plant nucleoskeleton and may have remained attached to proteins performing lamin-like functions 64 . With our data and the information about the Y-complex in humans, we were able to determine which elements of the Y-complex are present in both AtNPCs and HsNPCs and which ones are unique. We show that HsNPC and AtNPC share most NUPs of the Y-complex that are also conserved in other species such as Saccharomyces cerevisiae 66 . This highlights their important structural role in making up NPCs across different species. On the other hand, nucleoporins ELYS and NUP37, may have evolved to have specific functions in certain species. For example, it was previously reported that HOS1 (detected in several studies 38 , 67 – 71 ) contains a specific region with homology to ELYS. Based on this observation, it was proposed that HOS1 might be able to perform comparable functions to ELYS 38 . However, more functional information about ELYS and HOS1 is needed to determine whether they can fulfill similar functions in different organisms 72 . The techniques used to isolate the nuclear fraction, such as centrifugation, mechanical disruption, or exposure to certain buffers and reagents, could have disrupted the structural integrity of the NPC, leading to the detachment of some nucleoporins. While CG1 has been detected in other published studies 57 , this is likely due to the use of whole A. thaliana plantlet extracts for NUP detection, which enables the identification of proteins present across multiple tissue types or cell types. For Nup98b it was previously reported that isoforms a and b function redundantly and have a similar temporal expression pattern 63 , 73 thus if we detected unique peptides corresponding to NUP98a, we should have detected unique peptides corresponding to NUP98b. However, this apparent lack of detection of NUP98b is consistent with other proteomic analysis 40 . Although significant progress has been made in understanding the organization and composition of the NPCs in different organisms 10 , 11 , 13 , 14 , 22 , 24 , 59 , 74 , research on plants lags when compared to other eukaryotic species. Our study closes some of this gap, by obtaining the first structure of the NPC of a higher plant. Thus, we carry out a comprehensive comparison with NPCs from human but also with the more closely related single cell algae C. reinhardtii. This in-depth analysis enabled us to identify significant distinctions in the structure and dimensions of this large protein complex. Subcomplex segmentation and conserved integrative modeling based on predicted NUP structures and docking into the in situ cryo-ET map led to a comprehensive understanding of scaffold architecture. Our approach allowed for an insightful comparison between AtNPC, CrNPC and HsNPC. Our findings point out that the AtNPC has structural similarity with the CrNPC and is rather conserved with regards to the Y-complex arrangement and their distribution across CR and NR. Yet, NUP155 connector elements between the outer rings and the IR are prominent in the AtNPC structure and reminiscent of the connectors in HsNPC and absent in the CrNPC. The resemblance between the plant and human connector provides an interesting paradox suggesting that the last common eukaryotic ancestor (LECA) 75 would have had prominent connectors and that the shorter connectors, observed in Chlamydomonas , could have been acquired through divergent evolution. The AtNPCs in our protoplast cryo-ET dataset were present with a smaller membrane-to-membrane diameter as compared to previous HsNPC structure on isolated nuclear envelopes 14 , and which were known to be present in a constricted ground-state. In cells, several cryo-ET studies of different organisms have shown that NPCs reside in a more dilated state 16 , 76 , 77 . Constriction of NPCs have been previously linked to membrane tension, to energy depletion and hyperosmotic response as possible constriction cues 16 , 59 . While the smaller NPC diameter might be a true feature of higher plant cells in general, an alternative explanation could be that constriction of the in situ AtNPCs might result from lower membrane tension in root protoplasts or cell preparation. An alternative explanation might be the loss of shape and connective root tissue and thereby possibly decreasing tension. The small membrane-to-membrane diameter could also be specific to root cells, however, to date, there are no available protocols to perform this procedure from aerial organs. Despite these limitations, our approach reflects a powerful first demonstration of in situ structural biology directly in higher plants, leading to valuable new insights about the A. thaliana NPC complex and its structural conservation compared to unicellular algae and human cells. Our study opens the way for future structural biology studies from plant protoplast of different tissues to investigate other higher plant organelles and macromolecular complexes. Methods Plant Material and Growth Conditions Transgenic A. thaliana ecotype Columbia (Col-0) expressing green fluorescent protein (GFP) tagged to RNA export factor (RAE1) 38 were used in this study. Seeds were surface sterilized with 70% ethanol for 2 minutes, followed by 7% bleach and 0.1% Triton X-100 for 5 minutes. Then, seeds were washed 4–6 times with sterile water and stratified in the dark for 48 hours at 4ºC prior to germination. Seeds were sown at a density of approximately 50 seeds per Petri box over plant growth media consisting of ½ Murashige and Skoog Basal Medium (Sigma Aldrich) supplemented with 1% agar (Bio Basic) and adjusted to pH 5.7 with KOH ((Millipore Sigma). Square Petri dishes were positioned vertically under a long-day photoperiod (16 h of light, 8h of dark) at 23.5 ºC ± 0.5°C, with an average light intensity of 120 mmol/m 2 /s at the level of the rosette. Protoplast Isolation Protoplasts from roots were isolated according to Bargmann & Birnbaum (2010) 78 with minor modifications. Briefly, enzyme solution containing 0.4 M mannitol (Acros), 20 mM MES pH 5.7 (FisherBiotech), 20mM KCl (Fisher scientific), 1.5% Cellulase from Trichoderma sp . (Sigma-Aldrich), 0.3% Pectinase from Aspergillus niger (Sigma-Aldrich) was warmed at 55°C for 10 min, then cooled to room temperature. Then, 0.1% of (BSA), 10 mM of CaCl 2 (Bio Basic), and 5 mM of β-mercaptoethanol (Fisher Scientific) were added and the resulting solution was filtered through a 0.2 µm syringe filter (Fisher Scientific) into a Petri dish. Root tissue from 14-days-old plants was harvested with a scalpel and deposited and finely chopped in the petri dish containing the enzyme solution. The enzymatic digestion was performed under agitation at 75 rpm for 1.5 h at room temperature. After cell wall digestion, the solution was filtered through a 30 µm nylon mesh into a Falcon tube. One volume of W5 solution (154 mM NaCl (Fisher Scientific), 125 mM CaCl2 (Bio Basic), 5 mM KCl (Fisher Scientific), 2 mM MES pH 5.7 (FisherBiotech) was added, and protoplasts were spun for 10 min at 500 x g. Protoplasts were then resuspended in cold W5 solution and quantified under a light microscope and a Neubauer chamber to confirm their integrity. Then, protoplasts were also observed using a wide-field fluorescence microscope and 50 µL of Concanavalin A (concentration 1 mg/ml) to a clean glass-bottom dish to help immobilize the protoplasts while imaging them to assess GFP fluorescence and protoplast integrity. Nuclei Isolation Nuclei from root protoplasts were obtained according to the method described by Saxena, Fowke & King 37 but with some modifications. The whole procedure was carried out at 4°C. Briefly, cold Nuclear Isolation Buffer (NIB, 10mM MES.KOH (pH5.5), 0.2M sucrose, 2.5mM EDTA, 2.5mM DTT, 0.1mM spermine, 10mM NaCl, 10mM KCl, 0.15% Triton X-100) was added into a 50 ml round-bottomed centrifuge tube containing no more than 15 x 10 6 protoplasts per 15 mL. Deplasmolysis was enabled by keeping the NIB containing the protoplasts at 4°C for 7 minutes. The entire solution was then passed four times through a 26ga (brown) syringe needle. The solution containing the broken protoplasts was then passed through a 30 µm mesh. The filtered solution was centrifuged at 1000 x g for 8 minutes at 4°C. Then, a white pellet containing the nuclei was collected. For later usage, the nuclei were kept in aliquots in 1.5 ml Eppendorfs with NIB + 20% glycerol at -80 ºC. In total, three biological replicates were obtained for further analysis by mass spectrometry. Acetone precipitation Protein precipitation for mass spectrometry analysis was carried out based on a protocol provided by The Proteomics Platform of the Quebec Genomics Center at the CHU de Québec Research Center. Briefly, the samples containing the nuclear extracts were transferred to 2 mL Eppendorf tubes. To each of the samples 4 volumes of acetone were added at -20 ºC and then vortexed. Samples with acetone were incubated at -20 ºC overnight. Then, the samples were centrifuged at 16,000xg during 15 min at 4°C and the supernatant was discarded. The tubes containing the pellet with the nuclear extracts were dried under the hood for 5 minutes to let the acetone evaporate and finally the samples were resuspended in a protein extraction buffer containing 50 mM ammonium bicarbonate and 1% sodium deoxycholate. Mass spectrometry Nuclear extract samples were sent for analysis to detect the presence of NUPs to the Proteomics platform of the CHU de Québec Research Center. Using DTT (0.2 mM at 37°C for 30 minutes), iodoacetamide (0.8 mM at 37°C for 30 minutes), and trypsin (0.2 µg at a ratio of 1:50 protease/protein, 37°C overnight incubation), samples were reduced, alkylated, and digested, respectively. Tryptic peptides were desalted, vacuum dried and re-suspended in a 0.1% formic acid solution. Using the Nanodrop assay (205 nm absorbance), peptide quantities were calculated. A Dionex UltiMate 3000 nanoRSLC chromatography system (Thermo Fisher Scientific) coupled to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) was used for analyzing samples (1.0 ug) by nano LC/MSMS. Peptides were separated on a Pepmap Acclaim (ThermoFisher) 50 cm x 75 µm internal diameter separation column, using 300 nL/min for 90 minutes linear gradient from 5 to 40% solvent B (A: 0,1% formic acid, B: 80% acetonitrile, 0.1% formic acid). Thermo XCalibur software version 4.1.50 was used to collect mass spectra using a data dependent acquisition method. Precursor ions were analyzed in the Orbitrap at a resolution of 120 000 m/z and the most intense ions were selected for Higher-energy Collisional Dissociation (HCD) fragmentation by the quadrupole analyzer using 1.6 m/z isolation windows followed by fragments mass scans in the Ion Trap with a method set with a maximum cycle time of 3 seconds. Database searching MGF peak list files were generated by Proteome Discoverer 2.3 (Thermo Fisher Scientific). Filtered MS/MS data was then analyzed using Mascot (Matrix Science, London, UK; version 2.5.1). Mascot was set up to search against a UniProt A. thaliana Reference proteome database (UP000006548, version of August 24, 2020). Mascot search parameters included a 0.60 Da fragment ion mass tolerance and a 10.0 PPM parent ion tolerance. Cystein carbamidomethylation was set as a fixed modification while deamidation of asparagine and glutamine as well as the oxidation of methionine were set as variable modifications. Protein identification, and data analysis The Scaffold software (version 5.2.2, Proteomes Software Inc., Portland, USA) was used to validate MS/MS-based peptide and protein identification. Peptide identification was accepted if the Scaffold Local FDR method could establish it with higher than 91.0% probability to achieve an FDR less than 1.0%. Proteins that included at least two known peptides and could be identified with a probability of more than 99.0% to obtain an FDR less than 1.0% were acceptable. The Protein Prophet program assigns probability for each protein 79 . To adhere to the parsimony criteria, proteins with identical peptide compositions that could not be distinguished based only on MS/MS analysis were categorized. Data was considered using exponentially modified protein abundance index (emPAI) values 80 . Y-complex NUPs comparison All PDB AlphaFold files of NUPs from A. thaliana and H. sapiens were visualized using PyMOL Molecular Graphics System, Version 4.6 Schrödinger, LLC. TM-score alignment values TM-alignment for protein structure comparison was performed using the TM-align tool from the Zhang Lab group ( https://zhanggroup.org/TM-align/ ) 53 . Alphafold IDs and PDB files for the NUPs compared were obtained from the AlphaFold Protein Structure Database ( https://alphafold.ebi.ac.uk/ ). Cryo-EM sample preparation and Cryo-CLEM Cryo-EM grid preparation of root protoplasts for data acquisition were prepared as described earlier 26 . Briefly, using a Plunge Freezer (Leica GP2), set to 70% humidity, single backside blotting, 6 sec blot time, and no delay before blotting, EM grids (Au grids 200 mesh, SiO 2 foil, R2/1 from Quantifoil) were glow discharged two times with a Pelco easiGlow glow discharger for 90 s at 15 mA each. Root protoplasts were adjusted to a concentration of 500–650 protoplasts/µL in fresh W5 solution. A 3µL droplet containing 1500–1950 protoplasts was back-side blotted with Whatman filter paper grade 1 for 6 sec and vitrified onto each EM grid using a Leica Plunge Freezer EM GP2 by plunge freezing into liquid ethane. The frozen grids were clipped and imaged on an EM Cryo CLEM system (Leica Microsystems). Imaging was performed using a HC PL APO 50x/0.90 DRY objective, 488-nm laser excitation, and detecting simultaneously at 488–542 nm. Cryo-FIB milling Lamellae from plunge-frozen grids were prepared with an Aquilos FIB-SEM microscope (Thermo Scientific) similar to protocols that previously described this process 13 , 81 . Briefly, samples were coated with a layer of organometallic platinum for 10 s with the gas injection system (GIS). Then, they were additionally sputter coated with platinum for 10 s at voltage 1kV and 10 mA. Lamellae milling was performed in a step-wise fashion by decreasing the FIB current from 1 nA, 500 pA, 300 pA, to 100 pA. Final polishing of the lamellae was carried out with 30–50 pA to a final thickness of ~ 180–200 nm. Finally, an additional sputter layer of platinum at 1 kV and 10 mA was added for 1–2 s before unloading the sample. Cryo-ET acquisition A total of 111 tilt series were acquired in three independent microscope sessions on a Titan Krios G2 Cryo-Transmission Electron Microscope (Thermo Scientific), operating at 300 kV and equipped with a BioQuantum-K3 (Gatan) imaging filter. Before tilt series acquisition, the autogrids were carefully loaded with the lamella orientation perpendicular to the tilt axis of the microscope. Using SerialEM (version 3.8.1) in low dose mode, tilt series were recorded as 6 K x 4 K movies with 10 frames each, and motion-corrected in SerialEM on-the-fly. Projection images had a magnification of 42000x, corresponding to a unbinned pixel size of 2.176 Å. Acquisition of tilt series began either at 0° or with a lamella pretilt of -8°. A dose-symmetric acquisition strategy with 2° increments was used, yielding approximately 60 projections per tilt series with a constant exposure time, totaling about ~ 130 e-/2. The targeted defocus was adjusted from − 2.5 to − 5 µm, while the energy slit width was fixed at 20 eV. The detector dose rate was intended to be ~ 14.5 e–/px/s at lamella pretilt. Tilt-series processing All images were pre-processed by performing dose-filtering using MATLAB as described previously 82 . From dose-filtered tilt series, poor-quality tilt images were removed after visual inspection. Using the etomo program in IMOD 83 , dose-filtered tilt series were then aligned with the patch-tracking 83 , 84 and reconstructed as back-projected tomograms using the SIRT-like filtering at a binned pixel size of 8.704 Å. Based on the thickness of the reconstructed tomograms, quality of patch-tracking; and features contained in the volume 37 were chosen for further subtomogram averaging after visual assessment. For NPC subtomogram averaging, 3D CTF-corrected back-projected tomograms were generated using NovaCTF 85 . Subtomogram averaging A total of 79 NPCs were manually selected from 4-times binned tomograms. NPC coordinates and initial orientations were manually selected in tomograms that were filtered in a SIRT-like fashion 16 , 86 . Initial alignment of NPCs was carried out on the whole NPCs 16 , 50 . All alignment steps were performed using novaSTA 85 . The coordinates of NPC spokes were determined based on 8-fold symmetry after establishing an initial 4-times binned whole NPC map, as previously described 16 . A mask covering each asymmetric unit (CR, IR, and NR) was used for further alignment. Each subtomogram and its assigned orientation were manually examined following initial subunit alignment, and any misaligned or out of lamella particles were eliminated 87 , 88 . 4-times binned subtomograms and tight masks were used for focused alignment on the CR, IR, or NR. The final individual ring maps were first fitted to the STA map of the whole asymmetric subunit and a whole NPC composite map was created based on the 8-fold symmetry of the NPC. NPC diameter measurements Based on the final coordinates and orientations acquired during STA, NPC diameters were calculated using a MATLAB script that was already used in previous studies 16 . Only NPCs with a subunit occupancy of five or more were used when calculating the diameter of an NPC. Distances linking the opposing components were calculated for each individual NPC. The intersection point of all the vectors related to a certain NPC was used to establish the center of each NPC based on those distances. The typical NPC radius for a pore was determined to be the average distance between the newly found center and each individual subunit. This method allowed the evaluation of the average radius for a particular characteristic of interest inside each NPC. Structural modeling of the A. thaliana NUPs and NPC subcomplexes The structures of individual NUPs and NPC subcomplexes were modeled using AlphaFold2 51,89 available through AlphaPulldown 90 . The max_recycles parameter was set to 48. The following models were generated: NUP205-NUP93A (aa. 98–160), NUP205-NUP93B (aa. 98–160), NUP188-NUP93A (aa. 98–160), NUP188-NUP93B (aa. 98–160), NDC1-ALADIN, NUP54-NUP58-NUP62-NUP93A (aa. 1–95), NUP54-NUP58-NUP62-NUP93B (aa. 1–95), NUP93A (aa. 185–860)-NUP35 (aa. 1-150), NUP93B (aa. 185–860)-NUP35 (aa. 1-150), NUP35 (aa. 175–280)-NUP35 (aa. 175–280), NUP214 (aa. 730–950)-NUP88-NUP62 (aa. 540–739), NUP85-SEH1-NUP43, NUP160 (aa. 849–1495)-NUP85-SEH1, HOS1-NUP160-NUP96-SEC13A, NUP160-HOS1 (aa. 1-681)-NUP133 (aa. 577–1285), NUP96-SEC13A, NUP96-SEC13B, NUP96-NUP107, NUP160-NUP155 (aa. 1000–1464), NUP107-NUP133, NUP133 (aa. 577–1285)-NUP107-NUP93A (aa. 185–860), and NUP133 (aa. 577–1285)-NUP107-NUP93B (aa. 185–860) (Supplementary Fig. 1). Fitting of the AlphaFold models into cryo-ET maps To generate the model of the asymmetric unit of the A. thaliana NPC, we used the model of the human NPC (PDB 7R5J) 14 as templates. We fitted the IR and the fragments of CR and NR of the human NPC into the map of the A. thaliana NPC. Then, we superposed AlphaFold models of the A. thaliana NPC subcomplexes to the human model, and optimized the fits of the A. thaliana NPC subcomplexes into the map of the A. thalina NPC using ChimeraX 88 . Modeling of the A. thaliana NPC scaffold To assemble the model of the entire NPC scaffold we used the integrative modeling software Assembline 91 ,which is based on Integrative Modeling Platform (IMP) version 2.15 92 and Python Modeling Interface (PMI) 93 . In addition to using models of subcomplexes as rigid bodies for fitting in the modeling, several inter-subunit interfaces were restrained by elastic distance network derived from AlphaFold models, overlapping and bridging the already fitted models. During the refinement, the structures were used as rigid-bodies and simultaneously represented at two resolutions: Cα-only representation and a coarse-grained representation, in which 10-residue fragments were represented as a single bead. The Cα-only representation was used for all restraints except for the EM fit restraint. The NPC structure was optimized using the refinement protocol of Assembline to optimize the fit to the map, minimize steric clashes, and ensure connectivity of the protein linkers. The scoring function for the refinement comprised the EM fit restraint, clash score (SoftSpherePairScore of IMP), connectivity distance between domains neighboring in sequence, and elastic network restraints derived from the subcomplexes modeled with AlphaFold. Declarations Author contributions I.S. Conceptualization, analysis, investigation, methodology, writing, review, and editing original draft, funding acquisition. P.C.H. Conceptualization, analysis, investigation, methodology, writing, review, and editing original draft, funding acquisition. A.O.-K. Structural modeling, data analysis, writing original draft. V.F. Mass spectrometry analysis MB: Conceptualization, project administration, resources, supervision, writing, review, and editing. HG: Conceptualization, funding acquisition, resources, supervision, Writing, review, and editing. Acknowledgements We extend our appreciation to all the members of the Molecular Sociology Department (Max Planck, Frankfurt) for their guidance and input.and the Max Planck Computing and Data Facility for the computational resources. We thank Sonja Welsch, Mark Linder and the team from the Electron Microscopy Facility at the Max-Planck-Institute for Biophysics (Franckfurt) for their support, input and technical expertise. In Canada the research was supported by NSERC DG grant RG2020-04002 to HG. The internship of IBSC was supported by MITACS BRG grant and IBSC also received a PhD scholarship from the Fonds de Recherche du Québec en Nature et Technologie. This work was supported by the European Union (ERC, NPCvalve, project number 101054823 to M.B.). M.B. acknowledges funding by the Max Planck Society. P.C.H. was supported by an EMBO Postdoctoral Fellowship (ALTF 33-2021). References Boruc, J., Zhou, X. & Meier, I. Dynamics of the Plant Nuclear Envelope and Nuclear Pore. Plant Physiology 158 , 78-86 (2011). https://doi.org:10.1104/pp.111.185256 Raices, M. & D'Angelo, M. A. Nuclear pore complex composition: a new regulator of tissue-specific and developmental functions. Nature Reviews Molecular Cell Biology 13 , 687-699 (2012). https://doi.org:10.1038/nrm3461 Sun, J., Shi, Y. & Yildirim, E. The Nuclear Pore Complex in Cell Type-Specific Chromatin Structure and Gene Regulation. Trends in Genetics 35 , 579-588 (2019). https://doi.org:10.1016/j.tig.2019.05.006 Kramarz, K. et al. The nuclear pore primes recombination-dependent DNA synthesis at arrested forks by promoting SUMO removal. Nature Communications 11 , 5643 (2020). https://doi.org:10.1038/s41467-020-19516-z Strambio-De-Castillia, C., Niepel, M. & Rout, M. P. The nuclear pore complex: bridging nuclear transport and gene regulation. Nature Reviews Molecular Cell Biology 11 , 490-501 (2010). https://doi.org:10.1038/nrm2928 Meier, I., Richards, E. J. & Evans, D. E. Cell Biology of the Plant Nucleus. Annual Review of Plant Biology 68 , 139-172 (2017). https://doi.org:10.1146/annurev-arplant-042916-041115 Lin, D. H. & Hoelz, A. The structure of the nuclear pore complex (an update). Annual review of biochemistry 88 , 725-783 (2019). Hoelz, A., Debler, E. W. & Blobel, G. The structure of the nuclear pore complex. Annual review of biochemistry 80 , 613-643 (2011). von Appen, A. & Beck, M. Structure Determination of the Nuclear Pore Complex with Three-Dimensional Cryo electron Microscopy. Journal of Molecular Biology 428 , 2001-2010 (2016). https://doi.org:https://doi.org/10.1016/j.jmb.2016.01.004 Akey, C. W. et al. Comprehensive structure and functional adaptations of the yeast nuclear pore complex. Cell 185 , 361-378.e325 (2022). https://doi.org:10.1016/j.cell.2021.12.015 Beck, M. et al. Nuclear Pore Complex Structure and Dynamics Revealed by Cryoelectron Tomography. Science 306 , 1387-1390 (2004). https://doi.org:doi:10.1126/science.1104808 Eibauer, M. et al. Structure and gating of the nuclear pore complex. Nature Communications 6 , 7532 (2015). https://doi.org:10.1038/ncomms8532 Mosalaganti, S. et al. In situ architecture of the algal nuclear pore complex. Nature Communications 9 , 2361 (2018). https://doi.org:10.1038/s41467-018-04739-y Mosalaganti, S. et al. AI-based structure prediction empowers integrative structural analysis of human nuclear pores. Science 376 , eabm9506 (2022). https://doi.org:doi:10.1126/science.abm9506 Zhu, X. et al. Structure of the cytoplasmic ring of the Xenopus laevis nuclear pore complex. Science 376 , eabl8280 (2022). https://doi.org:10.1126/science.abl8280 Zimmerli, C. E. et al. Nuclear pores dilate and constrict in cellulo. Science 374 , eabd9776 (2021). https://doi.org:doi:10.1126/science.abd9776 Bley, C. J. et al. Architecture of the cytoplasmic face of the nuclear pore. Science 376 , eabm9129 (2022). https://doi.org:doi:10.1126/science.abm9129 Fontana, P. et al. Structure of cytoplasmic ring of nuclear pore complex by integrative cryo-EM and AlphaFold. Science 376 , eabm9326 (2022). https://doi.org:doi:10.1126/science.abm9326 Ibarra, A. & Hetzer, M. W. Nuclear pore proteins and the control of genome functions. Genes & Development 29 , 337-349 (2015). https://doi.org:10.1101/gad.256495.114 Kim, S. J. et al. Integrative structure and functional anatomy of a nuclear pore complex. Nature 555 , 475-482 (2018). https://doi.org:10.1038/nature26003 Yang, Q., Rout, M. P. & Akey, C. W. Three-Dimensional Architecture of the Isolated Yeast Nuclear Pore Complex: Functional and Evolutionary Implications. Molecular Cell 1 , 223-234 (1998). https://doi.org:10.1016/S1097-2765(00)80023-4 Huang, G. et al. Cryo-EM structure of the inner ring from the Xenopus laevis nuclear pore complex. Cell Research 32 , 451-460 (2022). https://doi.org:10.1038/s41422-022-00633-x Zhang, Y. et al. Molecular architecture of the luminal ring of the Xenopus laevis nuclear pore complex. Cell research 30 , 532-540 (2020). Zhu, X. et al. Structure of the cytoplasmic ring of the Xenopus laevis nuclear pore complex. Science 376 , eabl8280 (2022). Von Appen, A. et al. In situ structural analysis of the human nuclear pore complex. Nature 526 , 140-143 (2015). Sanchez Carrillo, I. B., Hoffmann, P. C., Barff, T., Beck, M. & Germain, H. Preparing Arabidopsis thaliana root protoplasts for cryo electron tomography. Frontiers in Plant Science 14 (2023). https://doi.org:10.3389/fpls.2023.1261180 Koornneef, M. & Meinke, D. The development of Arabidopsis as a model plant. The Plant Journal 61 , 909-921 (2010). https://doi.org:https://doi.org/10.1111/j.1365-313X.2009.04086.x Krämer, U. Planting molecular functions in an ecological context with Arabidopsis thaliana. eLife 4 , e06100 (2015). https://doi.org:10.7554/eLife.06100 Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408 , 796-815 (2000). https://doi.org:10.1038/35048692 Woodward, A. W. & Bartel, B. Biology in Bloom: A Primer on the Arabidopsis thaliana Model System. Genetics 208 , 1337-1349 (2018). https://doi.org:10.1534/genetics.118.300755 Davey, M. R., Anthony, P., Power, J. B. & Lowe, K. C. Plant protoplasts: status and biotechnological perspectives. Biotechnology Advances 23 , 131-171 (2005). https://doi.org:https://doi.org/10.1016/j.biotechadv.2004.09.008 Reyna-Llorens, I., Ferro-Costa, M. & Burgess, S. J. Plant protoplasts in the age of synthetic biology. Journal of Experimental Botany (2023). https://doi.org:10.1093/jxb/erad172 Shaw, R., Tian, X. & Xu, J. Single-Cell Transcriptome Analysis in Plants: Advances and Challenges. Molecular Plant 14 , 115-126 (2021). https://doi.org:10.1016/j.molp.2020.10.012 Sheen, J. Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol 127 , 1466-1475 (2001). Yoo, S.-D., Cho, Y.-H. & Sheen, J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocols 2 , 1565-1572 (2007). https://doi.org:10.1038/nprot.2007.199 Schaffer, M. et al. Optimized cryo-focused ion beam sample preparation aimed at in situ structural studies of membrane proteins. Journal of Structural Biology 197 , 73-82 (2017). https://doi.org:https://doi.org/10.1016/j.jsb.2016.07.010 Saxena, P. K., Fowke, L. C. & King, J. An efficient procedure for isolation of nuclei from plant protoplasts. Protoplasma 128 , 184-189 (1985). https://doi.org:10.1007/BF01276340 Tamura, K., Fukao, Y., Iwamoto, M., Haraguchi, T. & Hara-Nishimura, I. Identification and Characterization of Nuclear Pore Complex Components in Arabidopsis thaliana. The Plant Cell 22 , 4084 (2010). https://doi.org:10.1105/tpc.110.079947 Tamura, K. & Hara-Nishimura, I. The molecular architecture of the plant nuclear pore complex. Journal of Experimental Botany 64 , 823-832 (2012). https://doi.org:10.1093/jxb/ers258 Tang, Y., Huang, A. & Gu, Y. Global profiling of plant nuclear membrane proteome in Arabidopsis. Nature Plants 6 , 838-847 (2020). https://doi.org:10.1038/s41477-020-0700-9 Holzer, G. & Antonin, W. Breaking the Y. PLOS Genetics 15 , e1008109 (2019). https://doi.org:10.1371/journal.pgen.1008109 Asakawa, H. et al. Asymmetrical localization of Nup107-160 subcomplex components within the nuclear pore complex in fission yeast. PLOS Genetics 15 , e1008061 (2019). https://doi.org:10.1371/journal.pgen.1008061 Belgareh , N. m. et al. An evolutionarily conserved NPC subcomplex, which redistributes in part to kinetochores in mammalian cells. Journal of Cell Biology 154 , 1147-1160 (2001). https://doi.org:10.1083/jcb.200101081 Binder, A. & Parniske, M. Analysis of the Lotus japonicus nuclear pore NUP107-160 subcomplex reveals pronounced structural plasticity and functional redundancy. Frontiers in Plant Science 4 (2014). https://doi.org:10.3389/fpls.2013.00552 Kelley, K., Knockenhauer, K. E., Kabachinski, G. & Schwartz, T. U. Atomic structure of the Y complex of the nuclear pore. Nature Structural & Molecular Biology 22 , 425-431 (2015). https://doi.org:10.1038/nsmb.2998 Lutzmann, M., Kunze, R., Buerer, A., Aebi, U. & Hurt, E. Modular self-assembly of a Y-shaped multiprotein complex from seven nucleoporins. The EMBO Journal 21 , 387-397 (2002). https://doi.org:https://doi.org/10.1093/emboj/21.3.387 Stuwe, T. et al. Architecture of the nuclear pore complex coat. Science 347 , 1148-1152 (2015). https://doi.org:doi:10.1126/science.aaa4136 Walther, T. C. et al. The Conserved Nup107-160 Complex Is Critical for Nuclear Pore Complex Assembly. Cell 113 , 195-206 (2003). https://doi.org:10.1016/S0092-8674(03)00235-6 Wiermer, M. et al. Putative members of the Arabidopsis Nup107-160 nuclear pore sub-complex contribute to pathogen defense. The Plant Journal 70 , 796-808 (2012). https://doi.org:https://doi.org/10.1111/j.1365-313X.2012.04928.x Bui, Khanh H. et al. Integrated Structural Analysis of the Human Nuclear Pore Complex Scaffold. Cell 155 , 1233-1243 (2013). https://doi.org:10.1016/j.cell.2013.10.055 Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596 , 583-589 (2021). https://doi.org:10.1038/s41586-021-03819-2 Varadi, M. et al. AlphaFold Protein Structure Database in 2024: providing structure coverage for over 214 million protein sequences. Nucleic Acids Research 52 , D368-D375 (2023). https://doi.org:10.1093/nar/gkad1011 Zhang, Y. & Skolnick, J. TM-align: a protein structure alignment algorithm based on the TM-score. Nucleic Acids Research 33 , 2302-2309 (2005). https://doi.org:10.1093/nar/gki524 Faure, G. et al. iPBAvizu: a PyMOL plugin for an efficient 3D protein structure superimposition approach. Source Code for Biology and Medicine 14 , 5 (2019). https://doi.org:10.1186/s13029-019-0075-3 Varadi, M. et al. AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Research 50 , D439-D444 (2021). https://doi.org:10.1093/nar/gkab1061 Makarov, A. A., Padilla-Mejia, N. E. & Field, M. C. Evolution and diversification of the nuclear pore complex. Biochemical Society Transactions 49 , 1601-1619 (2021). https://doi.org:10.1042/bst20200570 Tang, Y. et al. Proxiome assembly of the plant nuclear pore reveals an essential hub for gene expression regulation. Nature Plants (2024). https://doi.org:10.1038/s41477-024-01698-9 Fernandez-Martinez, J. & Rout, M. P. One Ring to Rule them All? Structural and Functional Diversity in the Nuclear Pore Complex. Trends in Biochemical Sciences 46 , 595-607 (2021). https://doi.org:10.1016/j.tibs.2021.01.003 Hoffmann, P. C. et al. Nuclear pores as conduits for fluid flow during osmotic stress. bioRxiv , 2024.2001.2017.575985 (2024). https://doi.org:10.1101/2024.01.17.575985 Klughammer, N. et al. Diameter Dependence of Transport through Nuclear Pore Complex Mimics Studied Using Optical Nanopores. (2023). https://doi.org:10.1101/2023.02.18.529008 Gallemí, M. et al. DRACULA2 is a dynamic nucleoporin with a role in regulating the shade avoidance syndrome in Arabidopsis. Development 143 , 1623-1631 (2016). Ito, N. et al. Nuclear pore complex proteins are involved in centromere distribution. Iscience 27 (2024). Jiang, S. et al. Nucleoporin Nup98 participates in flowering regulation in a CONSTANS-independent mode. Plant Cell Reports 38 , 1263-1271 (2019). https://doi.org:10.1007/s00299-019-02442-w Mermet, S. et al. Evolutionarily conserved protein motifs drive interactions between the plant nucleoskeleton and nuclear pores. The Plant Cell 35 , 4284-4303 (2023). https://doi.org:10.1093/plcell/koad236 Neumann, N., Jeffares, D. C. & Poole, A. M. Outsourcing the nucleus: nuclear pore complex genes are no longer encoded in nucleomorph genomes. Evolutionary Bioinformatics 2 , 117693430600200023 (2006). Teimer, R., Kosinski, J., von Appen, A., Beck, M. & Hurt, E. A short linear motif in scaffold Nup145C connects Y-complex with pre-assembled outer ring Nup82 complex. Nature Communications 8 , 1107 (2017). https://doi.org:10.1038/s41467-017-01160-9 Dong, C.-H., Agarwal, M., Zhang, Y., Xie, Q. & Zhu, J.-K. The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1. Proceedings of the National Academy of Sciences 103 , 8281-8286 (2006). https://doi.org:doi:10.1073/pnas.0602874103 Han, S.-H., Park, Y.-J. & Park, C.-M. Publisher Correction: HOS1 activates DNA repair systems to enhance plant thermotolerance. Nature Plants 7 , 237-237 (2021). https://doi.org:10.1038/s41477-020-00842-5 Lee, K. & Seo, P. J. The Arabidopsis E3 ubiquitin ligase HOS1 contributes to auxin biosynthesis in the control of hypocotyl elongation. Plant Growth Regulation 76 , 157-165 (2015). https://doi.org:10.1007/s10725-014-9985-x Margalha, L. et al. HOS1 promotes plant tolerance to low-energy stress via the SnRK1 protein kinase. The Plant Journal 115 , 627-641 (2023). https://doi.org:https://doi.org/10.1111/tpj.16250 Shkryl, Y. et al. CRISPR/Cas9-Mediated Knockout of HOS1 Reveals Its Role in the Regulation of Secondary Metabolism in Arabidopsis thaliana. Plants 10 , 104 (2021). MacGregor, D. R. & Penfield, S. Exploring the pleiotropy of hos1. Journal of Experimental Botany 66 , 1661-1671 (2015). https://doi.org:10.1093/jxb/erv022 Xiao, L. et al. Two Nucleoporin98 homologous genes jointly participate in the regulation of starch degradation to repress senescence in Arabidopsis. BMC Plant Biology 20 , 292 (2020). https://doi.org:10.1186/s12870-020-02494-1 Beck, M., Lučić, V., Förster, F., Baumeister, W. & Medalia, O. Snapshots of nuclear pore complexes in action captured by cryo-electron tomography. Nature 449 , 611-615 (2007). https://doi.org:10.1038/nature06170 O’Malley, M. A., Leger, M. M., Wideman, J. G. & Ruiz-Trillo, I. Concepts of the last eukaryotic common ancestor. Nature Ecology & Evolution 3 , 338-344 (2019). https://doi.org:10.1038/s41559-019-0796-3 Schuller, A. P. et al. The cellular environment shapes the nuclear pore complex architecture. Nature 598 , 667-671 (2021). https://doi.org:10.1038/s41586-021-03985-3 Zila, V. et al. Cone-shaped HIV-1 capsids are transported through intact nuclear pores. Cell 184 , 1032-1046.e1018 (2021). https://doi.org:10.1016/j.cell.2021.01.025 Bargmann, B. O. & Birnbaum, K. D. Fluorescence activated cell sorting of plant protoplasts. J Vis Exp (2010). https://doi.org:10.3791/1673 Nesvizhskii, A. I., Keller, A., Kolker, E. & Aebersold, R. A statistical model for identifying proteins by tandem mass spectrometry. Analytical chemistry 75 , 4646-4658 (2003). Ishihama, Y. et al. Exponentially Modified Protein Abundance Index (emPAI) for Estimation of Absolute Protein Amount in Proteomics by the Number of Sequenced Peptides per Protein * S. Molecular & Cellular Proteomics 4 , 1265-1272 (2005). https://doi.org:10.1074/mcp.M500061-MCP200 Beck, M., Mosalaganti, S. & Kosinski, J. From the resolution revolution to evolution: structural insights into the evolutionary relationships between vesicle coats and the nuclear pore. Current Opinion in Structural Biology 52 , 32-40 (2018). https://doi.org:https://doi.org/10.1016/j.sbi.2018.07.012 Wan, W. et al. Structure and assembly of the Ebola virus nucleocapsid. Nature 551 , 394-397 (2017). https://doi.org:10.1038/nature24490 Mastronarde, D. N. & Held, S. R. Automated tilt series alignment and tomographic reconstruction in IMOD. Journal of Structural Biology 197 , 102-113 (2017). https://doi.org:https://doi.org/10.1016/j.jsb.2016.07.011 Hoffmann, P. C. et al. Structures of the eukaryotic ribosome and its translational states in situ. Nature Communications 13 , 7435 (2022). https://doi.org:10.1038/s41467-022-34997-w Turoňová, B., Schur, F. K. M., Wan, W. & Briggs, J. A. G. Efficient 3D-CTF correction for cryo-electron tomography using NovaCTF improves subtomogram averaging resolution to 3.4Å. Journal of Structural Biology 199 , 187-195 (2017). https://doi.org:https://doi.org/10.1016/j.jsb.2017.07.007 Allegretti, M. et al. In-cell architecture of the nuclear pore and snapshots of its turnover. Nature 586 , 796-800 (2020). https://doi.org:10.1038/s41586-020-2670-5 Ermel, U. H., Arghittu, S. M. & Frangakis, A. S. ArtiaX: An electron tomography toolbox for the interactive handling of sub-tomograms in UCSF ChimeraX. Protein Science 31 , e4472 (2022). https://doi.org:https://doi.org/10.1002/pro.4472 Goddard, T. D. et al. UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Science 27 , 14-25 (2018). https://doi.org:https://doi.org/10.1002/pro.3235 Evans, R. et al. Protein complex prediction with AlphaFold-Multimer. bioRxiv , 2021.2010.2004.463034 (2022). https://doi.org:10.1101/2021.10.04.463034 Yu, D., Chojnowski, G., Rosenthal, M. & Kosinski, J. AlphaPulldown—a python package for protein–protein interaction screens using AlphaFold-Multimer. Bioinformatics 39 (2022). https://doi.org:10.1093/bioinformatics/btac749 Rantos, V., Karius, K. & Kosinski, J. Integrative structural modeling of macromolecular complexes using Assembline. Nature Protocols 17 , 152-176 (2022). https://doi.org:10.1038/s41596-021-00640-z Webb, B. et al. Integrative structure modeling with the Integrative Modeling Platform. Protein Science 27 , 245-258 (2018). https://doi.org:https://doi.org/10.1002/pro.3311 Saltzberg, D. et al. in Biomolecular Simulations: Methods and Protocols (eds Massimiliano Bonomi & Carlo Camilloni) 353-377 (Springer New York, 2019). Additional Declarations There is NO Competing Interest. Supplementary Files Supplementarydata.docx Cite Share Download PDF Status: Published Journal Publication published 31 Oct, 2025 Read the published version in Nature Plants → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6197803","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":431058000,"identity":"b16fad6d-a290-4588-af01-d6fd478126f0","order_by":0,"name":"Hugo Germain","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYBACAwSTsYHhA8laGGcwMEiQooWBgZmHGC3mEukXHzD8ssnXbT/c9ti2zbrOvIH5IV4HWs7IKTZg7Euz3HYmsd04ty1dQuYAmzFeqwxu5KRJMPYcNjA7kNgmndt2WEKCgQe/66Ba/huYnX/YJm0J0cL8A7+W9GMSDD8OGJjdANrCCNHCht+WM2+YDRIbkoFaHrZJ9pxLl5zBzGZmgVfL8fSHDz78sQM6LP2ZxI8ya34J9ubHN/BpYWDgMWBIbIPzmMGIAGB/wMDwB1nLKBgFo2AUjAI0AAC2t0bwWlBPLwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-7046-6194","institution":"Université du Québec à Trois-Rivières","correspondingAuthor":true,"prefix":"","firstName":"Hugo","middleName":"","lastName":"Germain","suffix":""},{"id":431058001,"identity":"5e2a26ac-3f18-4970-b20d-c65a49e795d1","order_by":1,"name":"Ingrid Berenice Sanchez Carrillo","email":"","orcid":"","institution":"Université du Québec à Trois-Rivières","correspondingAuthor":false,"prefix":"","firstName":"Ingrid","middleName":"Berenice Sanchez","lastName":"Carrillo","suffix":""},{"id":431058002,"identity":"b6ef2c6b-f9a6-4b42-b52c-941018dc91b5","order_by":2,"name":"Patrick Hoffmann","email":"","orcid":"","institution":"Max-Planck-Institute for Biophysics","correspondingAuthor":false,"prefix":"","firstName":"Patrick","middleName":"","lastName":"Hoffmann","suffix":""},{"id":431058003,"identity":"e4aba85f-c164-4b38-9caa-cae8bdf6a349","order_by":3,"name":"Agnieszka Obarska-Kosinska","email":"","orcid":"","institution":"Max-Planck-Institute for Biophysics","correspondingAuthor":false,"prefix":"","firstName":"Agnieszka","middleName":"","lastName":"Obarska-Kosinska","suffix":""},{"id":431058004,"identity":"44445e47-3573-47db-b43c-465e5bfea4ec","order_by":4,"name":"Victor Fourcassié","email":"","orcid":"","institution":"Université Laval","correspondingAuthor":false,"prefix":"","firstName":"Victor","middleName":"","lastName":"Fourcassié","suffix":""},{"id":431058005,"identity":"c349e575-cbb0-4b1e-808a-874320652d8e","order_by":5,"name":"Martin Beck","email":"","orcid":"","institution":"Max-Planck-Institute for Biophysics","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"","lastName":"Beck","suffix":""}],"badges":[],"createdAt":"2025-03-10 18:21:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6197803/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6197803/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41477-025-02138-y","type":"published","date":"2025-10-31T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79065268,"identity":"322b2b91-15f2-439c-86a0-fbc940f6ddcd","added_by":"auto","created_at":"2025-03-24 04:05:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":998462,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 2 | Comparison of AlphaFold predicted structures of the NUPs that constitute the Y-complex in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. sapiens \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. thaliana.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eSide-by-side visualization of the AlphaFold-predicted tertiary structures of the NUPs that make up the Y-complex in both \u003cem\u003eA. thaliana \u003c/em\u003e(green) and \u003cem\u003eH. sapiens\u003c/em\u003e (blue). The characterized elements are represented as helices, beta sheets, and the uncharacterized regions are represented as lines. All files were downloaded from the AlphaFold Protein Structure Database (https://alphafold.ebi.ac.uk/), accessed and visualized with PyMOL.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6197803/v1/87c1ba36da832e7bd4d57dc0.png"},{"id":79065002,"identity":"b581cce2-c68f-43db-bc7e-9f40e390deef","added_by":"auto","created_at":"2025-03-24 03:57:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1014772,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 3 | Subtomogram average and structural model of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. thaliana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e NPC (AtNPC) from root protoplasts. (a) \u003c/strong\u003eSlice of a tomogram showing the NE with two embedded AtNPCs. (white arrowheads, scale bar: 100 nm) (\u003cstrong\u003eb)\u003c/strong\u003e STA composite map of the AtNPC resolved to ~35 Å for the individual rings. (\u003cstrong\u003ed) \u003c/strong\u003eSegmentation of the AtNPC from the cytosolic (top) and from the nuclear (bottom) view highlighting one Y-complex ring (orange) at the CR and two Y-complex rings at the NR (yellow and light blue), the NUP214 complex (yellow) and NUP205-NUP93 (dark blue). The IR spokes are shown in light green with the central channel facing NUP62 complexes shown in green. Connectors between both CR and NR to the IR are shown in dark green. (\u003cstrong\u003ed) \u003c/strong\u003eStructural model of the AtNPC built from predicted models of 20 NUPs, of which their presence was confirmed by mass spectrometry. Color code for NUPs is shown in the figure.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6197803/v1/7dd21d943d0a154d38f38393.png"},{"id":79065011,"identity":"0bcfac66-f35e-4a70-9ed1-9e9b5cac26aa","added_by":"auto","created_at":"2025-03-24 03:57:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":466974,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 4 | \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eIn situ\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e NPC scaffold architecture from \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. thaliana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e root protoplasts in comparison to the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. reinhardtii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eNPC (EMD-4355) and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. sapiens\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e NPC (EMD-14321). (a) \u003c/strong\u003eSide cut view of AtNPC, CrNPC, and HsNPC and diameter measured at the point of inner and outer nuclear membrane fusion as well as the height of the NPC scaffold. (\u003cstrong\u003eb)\u003c/strong\u003e Tilted views on the cytosolic side of the AtNPC, CrNPC, and HsNPC. All views are shown at the same scale \u003cstrong\u003eCR:\u003c/strong\u003ecytoplasmic ring, \u003cstrong\u003eIR: \u003c/strong\u003einner ring, and \u003cstrong\u003eNR:\u003c/strong\u003e nuclear ring\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6197803/v1/fbdccde60bedaaf1254ff370.png"},{"id":79065273,"identity":"195b8ddb-6a8b-449e-a10e-74f5c071405b","added_by":"auto","created_at":"2025-03-24 04:05:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":829473,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 5. Head-to-tail contact of the Y-complexes. \u003c/strong\u003e(a) The CR of the AtNPC. (b) The NR inner Y-complex of the AtNPC. The interface between NUP133 α-solenoid and HOS1 in (a) and (b) was predicted by AlphaFold2 (Supplementary Figure 1). (c) The NR outer Y-complex of the AtNPC. Density shown in pink may correspond to the NUP133 β-propeller, but it was not explicitly modeled since AlphaFold2 did not predict this interaction. (d) The inner Y-complex of the CR of the human NPC (PDB 7R5J) \u003csup\u003e14\u003c/sup\u003e. NUPs are colored as in Fig. 3.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6197803/v1/d31170463f7d1dc88d3a405e.png"},{"id":79065269,"identity":"c6b7c4ca-a02c-4bef-bef4-6e662bcd5682","added_by":"auto","created_at":"2025-03-24 04:05:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1016794,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 6.\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e A. thaliana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e NPC architecture. \u003c/strong\u003e(a) Zoom on the CR showing HOS1 (b) Zoom on the CR showing two copies of NUP205 and one copy of NUP93A/B. (c) Zoom on the NR showing two copies of HOS1. (d) Zoom on the NR showing NUP205 and the possible location of NUP93A/B. (e) AlphaFold models of the \u003cem\u003eA. thaliana \u003c/em\u003eNUP133-NUP107-NUP93A/B and the \u003cem\u003eH. sapiens\u003c/em\u003eNUP133-NUP107-NUP93 complexes (Supplementary Figure 1).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6197803/v1/6593ebd2bce04ea823891946.png"},{"id":94904497,"identity":"ff048964-2699-4028-a635-3870f205c047","added_by":"auto","created_at":"2025-11-01 07:11:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6147022,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6197803/v1/344ca5fb-9f3e-421b-bf22-bb5abc2c07f0.pdf"},{"id":79065270,"identity":"d42d0b51-3e13-4295-a483-b11eb07e4edf","added_by":"auto","created_at":"2025-03-24 04:05:36","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9667319,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-6197803/v1/2e69a7afbdf463c9affbffbc.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"In situ architecture of the nuclear pore complex of the higher plant Arabidopsis thaliana","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNuclear pore complexes (NPCs) are large multi-protein complexes involved in the selective import and export of macromolecules passing through the nuclear envelope (NE) \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Exclusive to eukaryotic organisms, NPCs play crucial roles in regulating gene expression \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, chromatin organization \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, DNA repair, and RNA processing and quality control \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. NPCs are organized into different subcomplexes \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e made of multiple copies (~\u0026thinsp;1000 protein subunits in humans \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e) of approximately 30 different proteins known as nucleoporins (NUPs) \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. With a few exceptions, NUPs are largely conserved among eukaryotes \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMuch of our understanding of the NPCs comes from various studies using electron microscopy (EM) \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, which has continued developing and refining, allowing the study of a broad range of model species \u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14 CR15\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Cryo electron tomography (cryo-ET) studies have revealed that the overall architecture of the NPC forms a three-layered scaffold structure\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, with an octagonal symmetry around the central transport channel\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. These three layers consist of the three main rings: the nuclear ring (NR), situated on the nucleoplasmic side; the inner ring (IR), embedded at the fusion point of the inner and outer nuclear membrane\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e ; and the cytoplasmic ring (CR). Structural data for the NPC of model organisms such as yeast\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eChlamydomonas\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, Xenopus \u003csup\u003e\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e and human \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e are now available, showcasing a range of structural differences within their NPC scaffolds. However, technical hurdles have limited the use of cryo-ET for higher plants and our understanding of plant NPCs largely relied on mass spectrometry \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and nucleoporin homology to better-characterized organisms. Recently we optimized the methodology to use cryo-ET to investigate the structural characteristics of protein complexes in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, which we use herein to provide insights into the evolution and origin of the plant NPC in eukaryotic organisms.\u003c/p\u003e \u003cp\u003e \u003cem\u003eA. thaliana\u003c/em\u003e is a small rosette plant that was first adopted as a model organism for its utility in genetic studies. It features a quick generation time, a small size (minimizing the need for extensive growing facilities), ease of genetic transformation, and prolific seed production through self-pollination. All of which make it a great model in plant biology \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In contrast to many organisms, \u003cem\u003eA. thaliana\u003c/em\u003e can withstand a high level of homozygosity and has a relatively small genome (132 Mbp) \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Despite its short life cycle, producing transgenic plants still requires several months \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. To circumvent these delays, electroporation and polyethylene glycol (PEG)-based transfection of protoplasts have grown in popularity as a tool for transient expression of genetic material. Since then, protoplasts have been extensively used to study various aspects of plant physiology, cell ultrastructure, and genetics \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Protoplasts are isolated cells without cell walls, and the enzymatic removal of the cell wall, known as protoplasting, does not obscure cell type differences or prevent comparisons with whole tissues, as it preserves physiological responses and cellular activities \u003csup\u003e\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. While procedures have been developed for employing protoplasts for various purposes, the use of \u003cem\u003ein situ\u003c/em\u003e cryo-ET to plant protoplasts was still limited until recently.\u003c/p\u003e \u003cp\u003eIn this study, we used a workflow using \u003cem\u003eA. thaliana\u003c/em\u003e root protoplasts, cryo focused ion beam (cryo-FIB) milling \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e of vitrified protoplasts for cryo-ET, and combined it with subtomogram averaging (STA) to examine and reveal the first NPC structure from a higher plant within its cellular environment. We constructed a model for the \u003cem\u003eA. thaliana\u003c/em\u003e NPC (AtNPC) based on the structure prediction of \u003cem\u003eA. thaliana\u003c/em\u003e nucleoporin homologs using integrative modeling. The identified scaffold arrangement is comparable to the previously published NPC of the unicellular algae \u003cem\u003eC. reinhardtii\u003c/em\u003e (CrNPC) \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, however with an overall smaller diameter than the \u003cem\u003eH. sapiens\u003c/em\u003e NPC (HsNPC) while maintaining a similar height and NUP155 connectors in the cytoplasmic ring.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMass spectrometry reveals thirty of the thirty-four NUPs of the plant NPC\u003c/h2\u003e \u003cp\u003eTo identify the NUPs that make up the \u003cem\u003eA. thaliana\u003c/em\u003e NPC we performed nuclear extraction \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e from root protoplasts followed with high-performance liquid chromatography and tandem mass spectrometry. Our results allowed us to identify thirty out of the thirty-four described \u003cem\u003eA. thaliana\u003c/em\u003e nucleoporins \u003csup\u003e\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e as being present in the root protoplasts (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) which is commendable considering that established protocols detect around 25 NUP \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Among the identified NUPs, we detected all NUPs of the inner ring, and most NUPs of the Y-complex. Only, NUP50a, NUP136, CG1, and NUP98b could not be detected with a false discovery rate (FDR) of 1%. NUP50a and NUP136 are usually found at the nuclear basket, while CG1 is located on the cytoplasmic side. NUP98b is a peripheral NUP located on both the cytoplasmic and nuclear sides of the central channel \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. A plausible explanation for the undetected NUPs, is that they may have become dissociated from the nuclear pore complex during the process of nuclear extraction and sample preparation. Our workflow demonstrated that we were able to detect most NUPs of the nuclear basket, nuclear and peripheral sides and use this information for further understanding of the elements that conform the plant NPC.\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\u003e\u003cb\u003eMass spectrometry identification of\u003c/b\u003e \u003cb\u003eA. thaliana\u003c/b\u003e \u003cb\u003eNUPs from nuclear extracts purified from\u003c/b\u003e \u003cb\u003eA. thaliana\u003c/b\u003e \u003cb\u003eroot protoplasts.\u003c/b\u003e List of \u003cem\u003eA. thaliana\u003c/em\u003e NUPs identified using mass spectrometry. The last column explains whether the proteins were detected or partially detected in the three technical replicates. Accession numbers were obtained from the Uniprot database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). empAI values were determined by carrying out a Mascot search (Matrix Science) and analysis with the Scaffold software (version 5.2.2, Proteomes Software Inc., Portland, USA).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\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=\"char\" char=\".\" 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=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAccession Number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eemPAI value Sample 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eemPAI value Sample 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eemPAI value Sample 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eStd deviation Sample 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eStd deviation Sample 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eStd deviation Sample 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePresence\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eALADIN\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAAAS_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.4000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.3560\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.1987\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.1267\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.1746\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0782\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNUP50C\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQ93ZH3_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0914\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0033\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHOS1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHOS1_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.5453\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.7713\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.6147\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0792\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.0665\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0990\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNUP205\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA0A1P8BGZ1_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.5220\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.5757\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.6167\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.1031\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.1561\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.1902\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGP210\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGP210_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9063\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.3200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.0507\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.3410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.0300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.1028\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNUP35\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNUP35_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.1230\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.1827\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0046\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.0809\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNUP43\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNUP43_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.1127\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.4867\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.2793\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.1115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.5187\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0588\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNUP50B\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNU50B_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0603\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.1045\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNUP54\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNUP54_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.1773\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.5193\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0601\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.1638\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNUP58\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNUP58_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.1572\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0867\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0182\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0782\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.0891\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0315\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNUP62\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNUP62_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.3720\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.6040\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0821\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.1005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.1459\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0037\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNUP85\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNUP85_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.6697\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.0183\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.6583\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.1230\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.2886\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.2657\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNUP88\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNUP88_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.1689\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.1087\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.1398\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0645\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.0586\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0632\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNUP93A\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNP93A_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.7823\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.6067\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.3477\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0926\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.3361\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.1736\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNUP93B\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNU93B_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.1094\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0654\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0948\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0777\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.0277\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0839\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNUP96\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNUP96_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.1643\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.3200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.1400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0609\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.1416\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0320\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNUP98A\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNU98A_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0542\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0764\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0189\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0235\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.0568\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0164\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNUP107\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNU107_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.1710\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.2847\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.2340\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0879\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.0081\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0187\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNUP133\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNU133_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.2017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.3237\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.2163\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0736\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.1061\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0097\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNUP155\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNU155_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.6537\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.7207\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.4023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0672\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.1554\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0668\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNUP160\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNU160_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.2690\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.2323\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.3240\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0501\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.1061\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0901\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNUP214\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNP214_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0080\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0155\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.0139\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0160\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNUA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNUA_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.4960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.6093\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.4250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.1025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.1793\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0267\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNUP188\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF4JUG3_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.1155\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.1032\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0177\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0360\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.0560\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0069\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNDC1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQ8LAF4_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.2993\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.1712\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.1066\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0589\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.0878\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0930\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGLE1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGLE1_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0135\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0233\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRAE1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRAE1_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.4657\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.0457\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.1330\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0692\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.9646\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0454\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSEH1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSEH1_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.4093\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.6237\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.2433\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.1678\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.2010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0663\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSEC13A\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSC13A_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.5030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.1993\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.1254\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.1828\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.0907\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.0595\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSEC13B\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSC13B_ARATH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.7567\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.9040\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.6103\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.5021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.2272\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.1463\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/3\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\u003e \u003cb\u003eStructural models of individual NUPs of the Y-complex are conserved between\u003c/b\u003e \u003cb\u003eA. thaliana\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eH. sapiens\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOf all the constituents that make up the NPC, the Y-complex (also known as the NUP84 complex in yeast or the NUP107\u0026ndash;Nup160 complex in vertebrates) is probably the best studied and structurally characterized NPC substructure \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Consisting of 6 to 10 NUPs depending on the organism \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, this complex is a main constituent of the cytoplasmic and nuclear ring \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Previous studies across different organisms \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan additionalcitationids=\"CR43 CR44 CR45 CR46 CR47 CR48\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e revealed the conservation of proteins and the architecture among eukaryotic species \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Based on the information from our mass spectrometry data, we compared the three-dimensional (3D) structures of the NUPs that make up the \u003cem\u003eA. thaliana\u003c/em\u003e Y-complex to visualize the structural similarities between the NUPs that make up their respective Y-complexes. To achieve this, we set side by side (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e) the AlphaFold predictions \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e of the NUPs from the Y-complex from \u003cem\u003eA. thaliana\u003c/em\u003e and \u003cem\u003eH. sapiens\u003c/em\u003e NUPs and measured the similarity between these NUPs (TM-score) \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e to assess their structural similarity. Comparing the 3D structures of proteins represents an opportunity to gain a deeper understanding of their evolutionary relationship \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eTM-score values of\u003c/b\u003e \u003cb\u003eA. thaliana\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eH.sapiens\u003c/b\u003e \u003cb\u003eNUPs from the Y-complex.\u003c/b\u003e TM-scores from \u003cem\u003eA. thaliana\u003c/em\u003e and \u003cem\u003eH.sapiens\u003c/em\u003e NUPs that are present in the Y-complex of both organisms using the TM-score tool from the Zhang group\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. ID accessions were obtained from the AlphaFold database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://alphafold.ebi.ac.uk/\u003c/span\u003e\u003cspan address=\"https://alphafold.ebi.ac.uk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e \u003csup\u003e51,52,55\u003c/sup\u003e ).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAlphaFold ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTM-score\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA.thaliana\u003c/em\u003e NUP160\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAF-F4IGA5-F1-v4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.58283\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH. sapiens\u003c/em\u003e NUP160\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAF-Q12769-F1-v4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA.thaliana\u003c/em\u003e NUP85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAF-Q8RXH2-F1-v4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.70345\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH. sapiens\u003c/em\u003e NUP85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAF-Q9BW27-F1-v4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA.thaliana\u003c/em\u003e NUP43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAF-Q24JJ9-F1-v4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.79516\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH. sapiens\u003c/em\u003e NUP43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAF-Q8NFH3-F1-v4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA.thaliana\u003c/em\u003e SEH1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAF-Q93VR9-F1-v4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.86101\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH. sapiens\u003c/em\u003e SEH1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAF-Q96EE3-F1-v4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA.thaliana\u003c/em\u003e NUP96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAF-Q8LLD0-F1-v4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.90428\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH. sapiens\u003c/em\u003e NUP96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAF-P52948-F1-v4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA.thaliana\u003c/em\u003e SEC13B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAF-O64740-F1-v4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.95452\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH. sapiens\u003c/em\u003e SEC13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAF-P55735-F1-v4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA.thaliana\u003c/em\u003e SEC13A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAF-Q9SRI1-F1-v4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.95315\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH. sapiens\u003c/em\u003e SEC13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAF-P55735-F1-v4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA.thaliana\u003c/em\u003e NUP107\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAF-Q8L748-F1-v4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.57271\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH. sapiens\u003c/em\u003e NUP107\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAF-P57740-F1-v4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA.thaliana\u003c/em\u003e NUP133\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAF-F4IGA5-F1-v4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.39414\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH. sapiens\u003c/em\u003e NUP133\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAF-Q8WUM0-F1-v4\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\u003eOur results confirmed that some components of the Y-complex that are found in vertebrates are broadly conserved among eukaryotic organisms as previously suggested \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. We found that the \u003cem\u003eA. thaliana\u003c/em\u003e Y-complex shares 8 out of 10 NUPs (NUP160, NUP85, NUP43, SEH1, NUP96, SEC13, NUP107, and NUP133) with that of \u003cem\u003eH. sapiens.\u003c/em\u003e The remaining two NUPs, NUP37 and ELYS, appear to be absent from the plant NPC as they have not been described from previous plant mass spectrometry experiments \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. However, based on sequence analysis the functional homolog of ELYS in plants is suspected to be HOS1 \u003csup\u003e38\u003c/sup\u003e. These findings suggest that, despite more than 500\u0026nbsp;million years of evolutionary separation between plant and animal cells, most of their Y-complex is conserved, with only a few species-specific nucleoporins.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe\u003c/b\u003e \u003cb\u003ein situ\u003c/b\u003e \u003cb\u003eNPC structure of higher plant cells revealed in\u003c/b\u003e \u003cb\u003eA. thaliana\u003c/b\u003e \u003cb\u003eroot protoplasts\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo study plant NPCs in their native state, we utilized a recently established workflow \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e employing isolated root protoplasts from transgenic \u003cem\u003eA. thaliana\u003c/em\u003e plants expressing RAE1-GFP \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e for \u003cem\u003ein situ\u003c/em\u003e cryo-ET. In brief, we vitrified the isolated protoplasts on cryo-EM grids and intact nuclei protoplasts expressing RAE1-GFP were then identified using a cryo Correlative Light and Electron Microscopy (cryo-CLEM). Protoplast lamellae were prepared using an Aquilos cryo-FIB microscope. We specifically targeted areas of the nuclear envelope for tilt-series acquisition in a cryo Transmission Electron Microscope (cryo-TEM), followed by tomogram reconstruction and subtomogram averaging (STA). These methods allowed us to structurally analyze the \u003cem\u003eA. thaliana\u003c/em\u003e NPC \u003cem\u003ein situ\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eWe acquired 37 tomograms of \u003cem\u003eA. thaliana\u003c/em\u003e nuclear envelopes which contained a total of 79 NPCs to examine the \u003cem\u003ein situ\u003c/em\u003e architecture of the plant NPC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Using a previously established subtomogram averaging approach \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e we obtained the cryo-ET map of the \u003cem\u003eA. thaliana\u003c/em\u003e NPC from these 79 NPCs at a resolution of 35\u0026Aring; for the focused maps of individual rings (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, c). To better understand the eight-fold symmetrical scaffold architecture, we segmented the cryo-ET map into subcomplexes. On the cytosolic side, the CR of the \u003cem\u003eA. thaliana\u003c/em\u003e NPC consists of one ring with eight copies of the Y-complex. In addition, each asymmetric unit contained density for the NUP214 complex and the NUP205-NUP93 complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). In contrast, the NR comprises two copies of the Y-complex complex per asymmetric unit and density for the NUP205-NUP93 complex, resulting in a total of 16 Y-complexes for the NR that form two concentric rings of inner and outer Y-complexes. Both CR and NR show prominent densities for NUP155 connectors linking them to the eight spokes of the IR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Having established the overall makeup of the \u003cem\u003eA. thaliana\u003c/em\u003e NPC, we constructed the first structural model of a higher plant NPC, based on our cryo-ET map, using predicted models \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e of 20 \u003cem\u003eA. thaliana\u003c/em\u003e NUPs, which we also previously had confirmed by mass spectrometry to be present in root protoplasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eA. thaliana\u003c/b\u003e \u003cb\u003eY-complex is comparable to\u003c/b\u003e \u003cb\u003eC. reinhardtii\u003c/b\u003e, \u003cb\u003ebut connector elements resemble those of\u003c/b\u003e \u003cb\u003eH. sapiens\u003c/b\u003e \u003cb\u003eNPC.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe individual subcomplexes comprising the CR, IR and NR of the NPCs are mostly conserved across different species \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Similarly, the \u003cem\u003eA. thaliana\u003c/em\u003e NPC subcomplexes exhibit high structural similarity to previously published NPC building blocks \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. To better illustrate the similarities of the \u003cem\u003eA. thaliana\u003c/em\u003e NPC scaffold and to highlight potential differences from the NPC structures of other species, we compared our \u003cem\u003eA. thaliana\u003c/em\u003e NPC (AtNPC) structure to the NPC of the single cell algae \u003cem\u003eC. reinhardtii\u003c/em\u003e (CrNPC) (EMD-4355 \u003csup\u003e13\u003c/sup\u003e) and the human \u003cem\u003ein situ\u003c/em\u003e NPC structure from HEK293 cells (HsNPC) (EMD-14321 \u003csup\u003e14\u003c/sup\u003e). Beyond the apparent similarities in the overall architecture, some striking contrasts are evident between the AtNPC, the CrNPC, and the HsNPC.\u003c/p\u003e \u003cp\u003eIn comparison to the HsNPC, which features an alternative Y-complex arrangement and two concentric rings for either the CR and NR, the AtNPC is generally shorter in height. Nevertheless, AtNPCs are taller than the CrNPC. The presence of the connectors elements (NUP155) between the outer rings and the IR spoke, which are not clearly resolved in the CrNPC\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, could contribute to this overall taller height (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). We also observe variations in the average diameter of the NPC (measured from membrane to membrane) across the three organisms. The AtNPC has the smallest average diameter measuring approximately 70 nm between the membranes, HsNPCs from HEK293 cells measure an average diameter of 92 nm in diameter and the CrNPCs have the largest average diameter with 102 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These differences could reflect different amounts of membrane tension under the given conditions between the three experiments. However, if these variations in diameter measurements and spatial dynamics reflect the architecture, they are likely to impact the function of nucleocytosolic transport as well as the osmotic response, possibly emphasizing the importance of understanding these differences \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe head-to-tail contact in the AtNPC CR Y-complex and NR inner Y-complex is formed between NUP133 α-solenoid domain and HOS1, while the head-to-tail contact in the human NPC is formed between NUP133 β-propeller domain and NUP160 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Fig. S1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eContrary to the human NPC, one copy of HOS1 appears present in the CR of the AtNPC while two HOS1 copies are present in the NR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). Moreover, two copies of a large nucleoporin likely representing NUP205 are in the CR of the AtNPC while only one copy is in the NR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, d).\u003c/p\u003e \u003cp\u003eThe C-terminal domain of NUP93A/B is positioned differently in the AtNPC compared to the C-terminal domain of NUP93 in the human NPC. There is no density in the AtNPC at the positions corresponding to NUP93 in the human NPC. The AlphaFold2 model of the interaction between NUP93/NUP93A/B, NUP133, and NUP107 looks different in human and \u003cem\u003eA. thaliana\u003c/em\u003e. NUP107 in \u003cem\u003eA. thaliana\u003c/em\u003e has an additional domain (not present in human NUP107) that interacts with NUP93A/B in \u003cem\u003eA. thaliana\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). The C-terminal domain of NUP93A/B seems to interact with NUP107 and NUP133 in the CR in the position modeled by AlphaFold2 (the density corresponding to NUP93A/B next to NUP107/NUP133). However, NUP93A/B is not present in the corresponding positions in the NR. There is another region in the NR that could possibly be occupied by NUP93A/B, but this interaction is not modeled by AlphaFold (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe presence of a double membrane layer separating the genetic material from the cytoplasm led to the formation of the nucleus, enabling nucleus-specific cellular processes. Direct passage through nuclear pores offers a great throughput, maintains semi-permeability and enables the controlling of molecular trafficking. The high degree of similarity among nuclear pores suggests they evolved from a common ancestor, later developing species-specific features. Despite the crucial importance of plants, technical limitations have hindered detailed investigation of their nuclear pore structure until recently. Here, we present the first detailed three-dimensional structure of the nuclear pore complex from a higher plant, providing insights into the evolutionary relationships that have shaped the NPC and its nucleoporins across and within kingdoms.\u003c/p\u003e \u003cp\u003eOur mass spectrometry results enabled us to compare the NUPs present in both \u003cem\u003eArabidopsis\u003c/em\u003e NPC with published data from human NPC. We identified thirty out of the thirty-four described \u003cem\u003eA. thaliana\u003c/em\u003e nucleoporins \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e as being present in the root protoplasts (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) suggesting that our nuclei purification protocol and mass spectrometry protocol were equally or more efficient in comparison to previously published protocol aiming NUP identification \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. The techniques used to isolate the nuclear fraction, such as centrifugation, mechanical disruption, and exposure to various buffers and reagents, might have disrupted the structural integrity of the NPC, leading to the detachment of the four missing nucleoporins. While these four NUPs have been detected in other published studies\u003csup\u003e38\u0026ndash;40,61\u0026minus;65\u003c/sup\u003e, this difference could be due to our exclusive use of root protoplast cells, whereas other studies utilized whole \u003cem\u003eA. thaliana\u003c/em\u003e plantlet, allowing for the detection of proteins present across multiple tissue or cell types. Specifically, for NUP136, it is known to be tethered to the plant nucleoskeleton and may have remained attached to proteins performing lamin-like functions\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. With our data and the information about the Y-complex in humans, we were able to determine which elements of the Y-complex are present in both AtNPCs and HsNPCs and which ones are unique. We show that HsNPC and AtNPC share most NUPs of the Y-complex that are also conserved in other species such as \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. This highlights their important structural role in making up NPCs across different species. On the other hand, nucleoporins ELYS and NUP37, may have evolved to have specific functions in certain species. For example, it was previously reported that HOS1 (detected in several studies \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan additionalcitationids=\"CR68 CR69 CR70\" citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e) contains a specific region with homology to ELYS. Based on this observation, it was proposed that HOS1 might be able to perform comparable functions to ELYS \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. However, more functional information about ELYS and HOS1 is needed to determine whether they can fulfill similar functions in different organisms \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe techniques used to isolate the nuclear fraction, such as centrifugation, mechanical disruption, or exposure to certain buffers and reagents, could have disrupted the structural integrity of the NPC, leading to the detachment of some nucleoporins. While CG1 has been detected in other published studies \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, this is likely due to the use of whole \u003cem\u003eA. thaliana\u003c/em\u003e plantlet extracts for NUP detection, which enables the identification of proteins present across multiple tissue types or cell types. For Nup98b it was previously reported that isoforms a and b function redundantly and have a similar temporal expression pattern\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e thus if we detected unique peptides corresponding to NUP98a, we should have detected unique peptides corresponding to NUP98b. However, this apparent lack of detection of NUP98b is consistent with other proteomic analysis \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlthough significant progress has been made in understanding the organization and composition of the NPCs in different organisms \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e, research on plants lags when compared to other eukaryotic species. Our study closes some of this gap, by obtaining the first structure of the NPC of a higher plant. Thus, we carry out a comprehensive comparison with NPCs from human but also with the more closely related single cell algae \u003cem\u003eC. reinhardtii.\u003c/em\u003e This in-depth analysis enabled us to identify significant distinctions in the structure and dimensions of this large protein complex. Subcomplex segmentation and conserved integrative modeling based on predicted NUP structures and docking into the in situ cryo-ET map led to a comprehensive understanding of scaffold architecture. Our approach allowed for an insightful comparison between AtNPC, CrNPC and HsNPC. Our findings point out that the AtNPC has structural similarity with the CrNPC and is rather conserved with regards to the Y-complex arrangement and their distribution across CR and NR. Yet, NUP155 connector elements between the outer rings and the IR are prominent in the AtNPC structure and reminiscent of the connectors in HsNPC and absent in the CrNPC. The resemblance between the plant and human connector provides an interesting paradox suggesting that the last common eukaryotic ancestor (LECA) \u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e would have had prominent connectors and that the shorter connectors, observed in \u003cem\u003eChlamydomonas\u003c/em\u003e, could have been acquired through divergent evolution.\u003c/p\u003e \u003cp\u003eThe AtNPCs in our protoplast cryo-ET dataset were present with a smaller membrane-to-membrane diameter as compared to previous HsNPC structure on isolated nuclear envelopes \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, and which were known to be present in a constricted ground-state. In cells, several cryo-ET studies of different organisms have shown that NPCs reside in a more dilated state \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e,\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. Constriction of NPCs have been previously linked to membrane tension, to energy depletion and hyperosmotic response as possible constriction cues \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. While the smaller NPC diameter might be a true feature of higher plant cells in general, an alternative explanation could be that constriction of the \u003cem\u003ein situ\u003c/em\u003e AtNPCs might result from lower membrane tension in root protoplasts or cell preparation. An alternative explanation might be the loss of shape and connective root tissue and thereby possibly decreasing tension. The small membrane-to-membrane diameter could also be specific to root cells, however, to date, there are no available protocols to perform this procedure from aerial organs.\u003c/p\u003e \u003cp\u003eDespite these limitations, our approach reflects a powerful first demonstration of \u003cem\u003ein situ\u003c/em\u003e structural biology directly in higher plants, leading to valuable new insights about the \u003cem\u003eA. thaliana\u003c/em\u003e NPC complex and its structural conservation compared to unicellular algae and human cells. Our study opens the way for future structural biology studies from plant protoplast of different tissues to investigate other higher plant organelles and macromolecular complexes.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePlant Material and Growth Conditions\u003c/h2\u003e \u003cp\u003eTransgenic \u003cem\u003eA. thaliana\u003c/em\u003e ecotype Columbia (Col-0) expressing green fluorescent protein (GFP) tagged to RNA export factor (RAE1)\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e were used in this study. Seeds were surface sterilized with 70% ethanol for 2 minutes, followed by 7% bleach and 0.1% Triton X-100 for 5 minutes. Then, seeds were washed 4\u0026ndash;6 times with sterile water and stratified in the dark for 48 hours at 4\u0026ordm;C prior to germination. Seeds were sown at a density of approximately 50 seeds per Petri box over plant growth media consisting of \u0026frac12; Murashige and Skoog Basal Medium (Sigma Aldrich) supplemented with 1% agar (Bio Basic) and adjusted to pH 5.7 with KOH ((Millipore Sigma). Square Petri dishes were positioned vertically under a long-day photoperiod (16 h of light, 8h of dark) at 23.5 \u0026ordm;C\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C, with an average light intensity of 120 mmol/m\u003csup\u003e2\u003c/sup\u003e/s at the level of the rosette.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eProtoplast Isolation\u003c/h3\u003e\n\u003cp\u003eProtoplasts from roots were isolated according to Bargmann \u0026amp; Birnbaum (2010) \u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e with minor modifications. Briefly, enzyme solution containing 0.4 M mannitol (Acros), 20 mM MES pH 5.7 (FisherBiotech), 20mM KCl (Fisher scientific), 1.5% Cellulase from \u003cem\u003eTrichoderma sp\u003c/em\u003e. (Sigma-Aldrich), 0.3% Pectinase from \u003cem\u003eAspergillus niger\u003c/em\u003e (Sigma-Aldrich) was warmed at 55\u0026deg;C for 10 min, then cooled to room temperature. Then, 0.1% of (BSA), 10 mM of CaCl\u003csub\u003e2\u003c/sub\u003e (Bio Basic), and 5 mM of β-mercaptoethanol (Fisher Scientific) were added and the resulting solution was filtered through a 0.2 \u0026micro;m syringe filter (Fisher Scientific) into a Petri dish. Root tissue from 14-days-old plants was harvested with a scalpel and deposited and finely chopped in the petri dish containing the enzyme solution. The enzymatic digestion was performed under agitation at 75 rpm for 1.5 h at room temperature. After cell wall digestion, the solution was filtered through a 30 \u0026micro;m nylon mesh into a Falcon tube. One volume of W5 solution (154 mM NaCl (Fisher Scientific), 125 mM CaCl2 (Bio Basic), 5 mM KCl (Fisher Scientific), 2 mM MES pH 5.7 (FisherBiotech) was added, and protoplasts were spun for 10 min at 500 x g. Protoplasts were then resuspended in cold W5 solution and quantified under a light microscope and a Neubauer chamber to confirm their integrity. Then, protoplasts were also observed using a wide-field fluorescence microscope and 50 \u0026micro;L of Concanavalin A (concentration 1 mg/ml) to a clean glass-bottom dish to help immobilize the protoplasts while imaging them to assess GFP fluorescence and protoplast integrity.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eNuclei Isolation\u003c/h2\u003e \u003cp\u003eNuclei from root protoplasts were obtained according to the method described by Saxena, Fowke \u0026amp; King \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e but with some modifications. The whole procedure was carried out at 4\u0026deg;C. Briefly, cold Nuclear Isolation Buffer (NIB, 10mM MES.KOH (pH5.5), 0.2M sucrose, 2.5mM EDTA, 2.5mM DTT, 0.1mM spermine, 10mM NaCl, 10mM KCl, 0.15% Triton X-100) was added into a 50 ml round-bottomed centrifuge tube containing no more than 15 x 10\u003csup\u003e6\u003c/sup\u003e protoplasts per 15 mL. Deplasmolysis was enabled by keeping the NIB containing the protoplasts at 4\u0026deg;C for 7 minutes. The entire solution was then passed four times through a 26ga (brown) syringe needle. The solution containing the broken protoplasts was then passed through a 30 \u0026micro;m mesh. The filtered solution was centrifuged at 1000 x g for 8 minutes at 4\u0026deg;C. Then, a white pellet containing the nuclei was collected. For later usage, the nuclei were kept in aliquots in 1.5 ml Eppendorfs with NIB\u0026thinsp;+\u0026thinsp;20% glycerol at -80 \u0026ordm;C. In total, three biological replicates were obtained for further analysis by mass spectrometry.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAcetone precipitation\u003c/h3\u003e\n\u003cp\u003eProtein precipitation for mass spectrometry analysis was carried out based on a protocol provided by The Proteomics Platform of the Quebec Genomics Center at the CHU de Qu\u0026eacute;bec Research Center. Briefly, the samples containing the nuclear extracts were transferred to 2 mL Eppendorf tubes. To each of the samples 4 volumes of acetone were added at -20 \u0026ordm;C and then vortexed. Samples with acetone were incubated at -20 \u0026ordm;C overnight. Then, the samples were centrifuged at 16,000xg during 15 min at 4\u0026deg;C and the supernatant was discarded. The tubes containing the pellet with the nuclear extracts were dried under the hood for 5 minutes to let the acetone evaporate and finally the samples were resuspended in a protein extraction buffer containing 50 mM ammonium bicarbonate and 1% sodium deoxycholate.\u003c/p\u003e\n\u003ch3\u003eMass spectrometry\u003c/h3\u003e\n\u003cp\u003eNuclear extract samples were sent for analysis to detect the presence of NUPs to the Proteomics platform of the CHU de Qu\u0026eacute;bec Research Center. Using DTT (0.2 mM at 37\u0026deg;C for 30 minutes), iodoacetamide (0.8 mM at 37\u0026deg;C for 30 minutes), and trypsin (0.2 \u0026micro;g at a ratio of 1:50 protease/protein, 37\u0026deg;C overnight incubation), samples were reduced, alkylated, and digested, respectively. Tryptic peptides were desalted, vacuum dried and re-suspended in a 0.1% formic acid solution. Using the Nanodrop assay (205 nm absorbance), peptide quantities were calculated. A Dionex UltiMate 3000 nanoRSLC chromatography system (Thermo Fisher Scientific) coupled to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) was used for analyzing samples (1.0 ug) by nano LC/MSMS. Peptides were separated on a Pepmap Acclaim (ThermoFisher) 50 cm x 75 \u0026micro;m internal diameter separation column, using 300 nL/min for 90 minutes linear gradient from 5 to 40% solvent B (A: 0,1% formic acid, B: 80% acetonitrile, 0.1% formic acid). Thermo XCalibur software version 4.1.50 was used to collect mass spectra using a data dependent acquisition method. Precursor ions were analyzed in the Orbitrap at a resolution of 120 000 m/z and the most intense ions were selected for Higher-energy Collisional Dissociation (HCD) fragmentation by the quadrupole analyzer using 1.6 m/z isolation windows followed by fragments mass scans in the Ion Trap with a method set with a maximum cycle time of 3 seconds.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDatabase searching\u003c/h2\u003e \u003cp\u003eMGF peak list files were generated by Proteome Discoverer 2.3 (Thermo Fisher Scientific). Filtered MS/MS data was then analyzed using Mascot (Matrix Science, London, UK; version 2.5.1). Mascot was set up to search against a UniProt \u003cem\u003eA. thaliana\u003c/em\u003e Reference proteome database (UP000006548, version of August 24, 2020). Mascot search parameters included a 0.60 Da fragment ion mass tolerance and a 10.0 PPM parent ion tolerance. Cystein carbamidomethylation was set as a fixed modification while deamidation of asparagine and glutamine as well as the oxidation of methionine were set as variable modifications.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eProtein identification, and data analysis\u003c/h2\u003e \u003cp\u003eThe Scaffold software (version 5.2.2, Proteomes Software Inc., Portland, USA) was used to validate MS/MS-based peptide and protein identification. Peptide identification was accepted if the Scaffold Local FDR method could establish it with higher than 91.0% probability to achieve an FDR less than 1.0%. Proteins that included at least two known peptides and could be identified with a probability of more than 99.0% to obtain an FDR less than 1.0% were acceptable. The Protein Prophet program assigns probability for each protein \u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e. To adhere to the parsimony criteria, proteins with identical peptide compositions that could not be distinguished based only on MS/MS analysis were categorized. Data was considered using exponentially modified protein abundance index (emPAI) values \u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eY-complex NUPs comparison\u003c/h2\u003e \u003cp\u003eAll PDB AlphaFold files of NUPs from \u003cem\u003eA. thaliana\u003c/em\u003e and \u003cem\u003eH. sapiens\u003c/em\u003e were visualized using PyMOL Molecular Graphics System, Version 4.6 Schr\u0026ouml;dinger, LLC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTM-score alignment values\u003c/h2\u003e \u003cp\u003eTM-alignment for protein structure comparison was performed using the TM-align tool from the Zhang Lab group (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://zhanggroup.org/TM-align/\u003c/span\u003e\u003cspan address=\"https://zhanggroup.org/TM-align/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Alphafold IDs and PDB files for the NUPs compared were obtained from the AlphaFold Protein Structure Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://alphafold.ebi.ac.uk/\u003c/span\u003e\u003cspan address=\"https://alphafold.ebi.ac.uk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCryo-EM sample preparation and Cryo-CLEM\u003c/h2\u003e \u003cp\u003eCryo-EM grid preparation of root protoplasts for data acquisition were prepared as described earlier \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Briefly, using a Plunge Freezer (Leica GP2), set to 70% humidity, single backside blotting, 6 sec blot time, and no delay before blotting, EM grids (Au grids 200 mesh, SiO\u003csub\u003e2\u003c/sub\u003e foil, R2/1 from Quantifoil) were glow discharged two times with a Pelco easiGlow glow discharger for 90 s at 15 mA each. Root protoplasts were adjusted to a concentration of 500\u0026ndash;650 protoplasts/\u0026micro;L in fresh W5 solution. A 3\u0026micro;L droplet containing 1500\u0026ndash;1950 protoplasts was back-side blotted with Whatman filter paper grade 1 for 6 sec and vitrified onto each EM grid using a Leica Plunge Freezer EM GP2 by plunge freezing into liquid ethane. The frozen grids were clipped and imaged on an EM Cryo CLEM system (Leica Microsystems). Imaging was performed using a HC PL APO 50x/0.90 DRY objective, 488-nm laser excitation, and detecting simultaneously at 488\u0026ndash;542 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCryo-FIB milling\u003c/h2\u003e \u003cp\u003eLamellae from plunge-frozen grids were prepared with an Aquilos FIB-SEM microscope (Thermo Scientific) similar to protocols that previously described this process \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e. Briefly, samples were coated with a layer of organometallic platinum for 10 s with the gas injection system (GIS). Then, they were additionally sputter coated with platinum for 10 s at voltage 1kV and 10 mA. Lamellae milling was performed in a step-wise fashion by decreasing the FIB current from 1 nA, 500 pA, 300 pA, to 100 pA. Final polishing of the lamellae was carried out with 30\u0026ndash;50 pA to a final thickness of ~\u0026thinsp;180\u0026ndash;200 nm. Finally, an additional sputter layer of platinum at 1 kV and 10 mA was added for 1\u0026ndash;2 s before unloading the sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCryo-ET acquisition\u003c/h2\u003e \u003cp\u003eA total of 111 tilt series were acquired in three independent microscope sessions on a Titan Krios G2 Cryo-Transmission Electron Microscope (Thermo Scientific), operating at 300 kV and equipped with a BioQuantum-K3 (Gatan) imaging filter. Before tilt series acquisition, the autogrids were carefully loaded with the lamella orientation perpendicular to the tilt axis of the microscope. Using SerialEM (version 3.8.1) in low dose mode, tilt series were recorded as 6 K x 4 K movies with 10 frames each, and motion-corrected in SerialEM on-the-fly. Projection images had a magnification of 42000x, corresponding to a unbinned pixel size of 2.176 \u0026Aring;. Acquisition of tilt series began either at 0\u0026deg; or with a lamella pretilt of -8\u0026deg;. A dose-symmetric acquisition strategy with 2\u0026deg; increments was used, yielding approximately 60 projections per tilt series with a constant exposure time, totaling about\u0026thinsp;~\u0026thinsp;130 e-/2. The targeted defocus was adjusted from \u0026minus;\u0026thinsp;2.5 to \u0026minus;\u0026thinsp;5 \u0026micro;m, while the energy slit width was fixed at 20 eV. The detector dose rate was intended to be ~\u0026thinsp;14.5 e\u0026ndash;/px/s at lamella pretilt.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eTilt-series processing\u003c/h2\u003e \u003cp\u003eAll images were pre-processed by performing dose-filtering using MATLAB as described previously \u003csup\u003e\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e. From dose-filtered tilt series, poor-quality tilt images were removed after visual inspection. Using the etomo program in IMOD \u003csup\u003e\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e, dose-filtered tilt series were then aligned with the patch-tracking \u003csup\u003e\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e,\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e and reconstructed as back-projected tomograms using the SIRT-like filtering at a binned pixel size of 8.704 \u0026Aring;. Based on the thickness of the reconstructed tomograms, quality of patch-tracking; and features contained in the volume 37 were chosen for further subtomogram averaging after visual assessment. For NPC subtomogram averaging, 3D CTF-corrected back-projected tomograms were generated using NovaCTF \u003csup\u003e\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSubtomogram averaging\u003c/h2\u003e \u003cp\u003eA total of 79 NPCs were manually selected from 4-times binned tomograms. NPC coordinates and initial orientations were manually selected in tomograms that were filtered in a SIRT-like fashion \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u003c/sup\u003e. Initial alignment of NPCs was carried out on the whole NPCs \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. All alignment steps were performed using novaSTA\u003csup\u003e\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e. The coordinates of NPC spokes were determined based on 8-fold symmetry after establishing an initial 4-times binned whole NPC map, as previously described \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. A mask covering each asymmetric unit (CR, IR, and NR) was used for further alignment. Each subtomogram and its assigned orientation were manually examined following initial subunit alignment, and any misaligned or out of lamella particles were eliminated \u003csup\u003e\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e,\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e. 4-times binned subtomograms and tight masks were used for focused alignment on the CR, IR, or NR. The final individual ring maps were first fitted to the STA map of the whole asymmetric subunit and a whole NPC composite map was created based on the 8-fold symmetry of the NPC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eNPC diameter measurements\u003c/h2\u003e \u003cp\u003eBased on the final coordinates and orientations acquired during STA, NPC diameters were calculated using a MATLAB script that was already used in previous studies \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Only NPCs with a subunit occupancy of five or more were used when calculating the diameter of an NPC. Distances linking the opposing components were calculated for each individual NPC. The intersection point of all the vectors related to a certain NPC was used to establish the center of each NPC based on those distances. The typical NPC radius for a pore was determined to be the average distance between the newly found center and each individual subunit. This method allowed the evaluation of the average radius for a particular characteristic of interest inside each NPC.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStructural modeling of the\u003c/b\u003e \u003cb\u003eA. thaliana\u003c/b\u003e \u003cb\u003eNUPs and NPC subcomplexes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe structures of individual NUPs and NPC subcomplexes were modeled using AlphaFold2 \u003csup\u003e51,89\u003c/sup\u003e available through AlphaPulldown \u003csup\u003e\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e\u003c/sup\u003e. The max_recycles parameter was set to 48. The following models were generated: NUP205-NUP93A (aa. 98\u0026ndash;160), NUP205-NUP93B (aa. 98\u0026ndash;160), NUP188-NUP93A (aa. 98\u0026ndash;160), NUP188-NUP93B (aa. 98\u0026ndash;160), NDC1-ALADIN, NUP54-NUP58-NUP62-NUP93A (aa. 1\u0026ndash;95), NUP54-NUP58-NUP62-NUP93B (aa. 1\u0026ndash;95), NUP93A (aa. 185\u0026ndash;860)-NUP35 (aa. 1-150), NUP93B (aa. 185\u0026ndash;860)-NUP35 (aa. 1-150), NUP35 (aa. 175\u0026ndash;280)-NUP35 (aa. 175\u0026ndash;280), NUP214 (aa. 730\u0026ndash;950)-NUP88-NUP62 (aa. 540\u0026ndash;739), NUP85-SEH1-NUP43, NUP160 (aa. 849\u0026ndash;1495)-NUP85-SEH1, HOS1-NUP160-NUP96-SEC13A, NUP160-HOS1 (aa. 1-681)-NUP133 (aa. 577\u0026ndash;1285), NUP96-SEC13A, NUP96-SEC13B, NUP96-NUP107, NUP160-NUP155 (aa. 1000\u0026ndash;1464), NUP107-NUP133, NUP133 (aa. 577\u0026ndash;1285)-NUP107-NUP93A (aa. 185\u0026ndash;860), and NUP133 (aa. 577\u0026ndash;1285)-NUP107-NUP93B (aa. 185\u0026ndash;860) (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eFitting of the AlphaFold models into cryo-ET maps\u003c/h2\u003e \u003cp\u003eTo generate the model of the asymmetric unit of the \u003cem\u003eA. thaliana\u003c/em\u003e NPC, we used the model of the human NPC (PDB 7R5J) \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e as templates. We fitted the IR and the fragments of CR and NR of the human NPC into the map of the A. \u003cem\u003ethaliana\u003c/em\u003e NPC. Then, we superposed AlphaFold models of the A. \u003cem\u003ethaliana\u003c/em\u003e NPC subcomplexes to the human model, and optimized the fits of the A. \u003cem\u003ethaliana\u003c/em\u003e NPC subcomplexes into the map of the A. \u003cem\u003ethalina\u003c/em\u003e NPC using ChimeraX \u003csup\u003e\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eModeling of the\u003c/b\u003e \u003cb\u003eA. thaliana\u003c/b\u003e \u003cb\u003eNPC scaffold\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo assemble the model of the entire NPC scaffold we used the integrative modeling software Assembline \u003csup\u003e\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e\u003c/sup\u003e,which is based on Integrative Modeling Platform (IMP) version 2.15 \u003csup\u003e92\u003c/sup\u003e and Python Modeling Interface (PMI) \u003csup\u003e\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition to using models of subcomplexes as rigid bodies for fitting in the modeling, several inter-subunit interfaces were restrained by elastic distance network derived from AlphaFold models, overlapping and bridging the already fitted models. During the refinement, the structures were used as rigid-bodies and simultaneously represented at two resolutions: Cα-only representation and a coarse-grained representation, in which 10-residue fragments were represented as a single bead. The Cα-only representation was used for all restraints except for the EM fit restraint.\u003c/p\u003e \u003cp\u003eThe NPC structure was optimized using the refinement protocol of Assembline to optimize the fit to the map, minimize steric clashes, and ensure connectivity of the protein linkers. The scoring function for the refinement comprised the EM fit restraint, clash score (SoftSpherePairScore of IMP), connectivity distance between domains neighboring in sequence, and elastic network restraints derived from the subcomplexes modeled with AlphaFold.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI.S. Conceptualization, analysis, investigation, methodology, writing, review, and editing original draft, funding acquisition. P.C.H. Conceptualization, analysis, investigation, methodology, writing, review, and editing original draft, funding acquisition. A.O.-K. Structural modeling, data analysis, writing original draft. V.F. Mass spectrometry analysis MB: Conceptualization, project administration, resources, supervision, writing, review, and editing. HG: Conceptualization, funding acquisition, resources, supervision, Writing, review, and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our appreciation to all the members of the Molecular Sociology Department (Max Planck, Frankfurt) for their guidance and input.and the Max Planck Computing and Data Facility for the computational\u0026nbsp;resources. We thank Sonja Welsch, Mark Linder and the team from the Electron Microscopy Facility at the Max-Planck-Institute for Biophysics (Franckfurt) for their support, input and technical expertise. In Canada the research was supported by NSERC DG grant RG2020-04002 to HG. The internship of IBSC was supported by MITACS BRG grant and IBSC also received a PhD scholarship from the Fonds de Recherche du Québec en Nature et Technologie.\u0026nbsp;This work was supported by the European Union (ERC, NPCvalve, project number 101054823 to M.B.).\u0026nbsp;M.B. acknowledges funding by the Max Planck Society. P.C.H. was supported by an EMBO Postdoctoral Fellowship (ALTF 33-2021).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBoruc, J., Zhou, X. \u0026amp; Meier, I. Dynamics of the Plant Nuclear Envelope and Nuclear Pore. \u003cem\u003ePlant Physiology\u003c/em\u003e \u003cstrong\u003e158\u003c/strong\u003e, 78-86 (2011). https://doi.org:10.1104/pp.111.185256\u003c/li\u003e\n \u003cli\u003eRaices, M. \u0026amp; D\u0026apos;Angelo, M. A. Nuclear pore complex composition: a new regulator of tissue-specific and developmental functions. \u003cem\u003eNature Reviews Molecular Cell Biology\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 687-699 (2012). https://doi.org:10.1038/nrm3461\u003c/li\u003e\n \u003cli\u003eSun, J., Shi, Y. \u0026amp; Yildirim, E. The Nuclear Pore Complex in Cell Type-Specific Chromatin Structure and Gene Regulation. \u003cem\u003eTrends in Genetics\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 579-588 (2019). https://doi.org:10.1016/j.tig.2019.05.006\u003c/li\u003e\n \u003cli\u003eKramarz, K.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e The nuclear pore primes recombination-dependent DNA synthesis at arrested forks by promoting SUMO removal. \u003cem\u003eNature Communications\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 5643 (2020). https://doi.org:10.1038/s41467-020-19516-z\u003c/li\u003e\n \u003cli\u003eStrambio-De-Castillia, C., Niepel, M. \u0026amp; Rout, M. P. The nuclear pore complex: bridging nuclear transport and gene regulation. \u003cem\u003eNature Reviews Molecular Cell Biology\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 490-501 (2010). https://doi.org:10.1038/nrm2928\u003c/li\u003e\n \u003cli\u003eMeier, I., Richards, E. J. \u0026amp; Evans, D. E. Cell Biology of the Plant Nucleus. \u003cem\u003eAnnual Review of Plant Biology\u003c/em\u003e \u003cstrong\u003e68\u003c/strong\u003e, 139-172 (2017). https://doi.org:10.1146/annurev-arplant-042916-041115\u003c/li\u003e\n \u003cli\u003eLin, D. H. \u0026amp; Hoelz, A. The structure of the nuclear pore complex (an update). \u003cem\u003eAnnual review of biochemistry\u003c/em\u003e \u003cstrong\u003e88\u003c/strong\u003e, 725-783 (2019).\u003c/li\u003e\n \u003cli\u003eHoelz, A., Debler, E. W. \u0026amp; Blobel, G. The structure of the nuclear pore complex. \u003cem\u003eAnnual review of biochemistry\u003c/em\u003e \u003cstrong\u003e80\u003c/strong\u003e, 613-643 (2011).\u003c/li\u003e\n \u003cli\u003evon Appen, A. \u0026amp; Beck, M. Structure Determination of the Nuclear Pore Complex with Three-Dimensional Cryo electron Microscopy. \u003cem\u003eJournal of Molecular Biology\u003c/em\u003e \u003cstrong\u003e428\u003c/strong\u003e, 2001-2010 (2016). https://doi.org:https://doi.org/10.1016/j.jmb.2016.01.004\u003c/li\u003e\n \u003cli\u003eAkey, C. W.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Comprehensive structure and functional adaptations of the yeast nuclear pore complex. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e185\u003c/strong\u003e, 361-378.e325 (2022). https://doi.org:10.1016/j.cell.2021.12.015\u003c/li\u003e\n \u003cli\u003eBeck, M.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Nuclear Pore Complex Structure and Dynamics Revealed by Cryoelectron Tomography. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e306\u003c/strong\u003e, 1387-1390 (2004). https://doi.org:doi:10.1126/science.1104808\u003c/li\u003e\n \u003cli\u003eEibauer, M.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Structure and gating of the nuclear pore complex. \u003cem\u003eNature Communications\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 7532 (2015). https://doi.org:10.1038/ncomms8532\u003c/li\u003e\n \u003cli\u003eMosalaganti, S.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e In situ architecture of the algal nuclear pore complex. \u003cem\u003eNature Communications\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 2361 (2018). https://doi.org:10.1038/s41467-018-04739-y\u003c/li\u003e\n \u003cli\u003eMosalaganti, S.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e AI-based structure prediction empowers integrative structural analysis of human nuclear pores. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e376\u003c/strong\u003e, eabm9506 (2022). https://doi.org:doi:10.1126/science.abm9506\u003c/li\u003e\n \u003cli\u003eZhu, X.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Structure of the cytoplasmic ring of the Xenopus laevis nuclear pore complex. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e376\u003c/strong\u003e, eabl8280 (2022). https://doi.org:10.1126/science.abl8280\u003c/li\u003e\n \u003cli\u003eZimmerli, C. E.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Nuclear pores dilate and constrict in cellulo. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e374\u003c/strong\u003e, eabd9776 (2021). https://doi.org:doi:10.1126/science.abd9776\u003c/li\u003e\n \u003cli\u003eBley, C. J.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Architecture of the cytoplasmic face of the nuclear pore. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e376\u003c/strong\u003e, eabm9129 (2022). https://doi.org:doi:10.1126/science.abm9129\u003c/li\u003e\n \u003cli\u003eFontana, P.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Structure of cytoplasmic ring of nuclear pore complex by integrative cryo-EM and AlphaFold. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e376\u003c/strong\u003e, eabm9326 (2022). https://doi.org:doi:10.1126/science.abm9326\u003c/li\u003e\n \u003cli\u003eIbarra, A. \u0026amp; Hetzer, M. W. Nuclear pore proteins and the control of genome functions. \u003cem\u003eGenes \u0026amp; Development\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 337-349 (2015). https://doi.org:10.1101/gad.256495.114\u003c/li\u003e\n \u003cli\u003eKim, S. J.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Integrative structure and functional anatomy of a nuclear pore complex. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e555\u003c/strong\u003e, 475-482 (2018). https://doi.org:10.1038/nature26003\u003c/li\u003e\n \u003cli\u003eYang, Q., Rout, M. P. \u0026amp; Akey, C. W. Three-Dimensional Architecture of the Isolated Yeast Nuclear Pore Complex: Functional and Evolutionary Implications. \u003cem\u003eMolecular Cell\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 223-234 (1998). https://doi.org:10.1016/S1097-2765(00)80023-4\u003c/li\u003e\n \u003cli\u003eHuang, G.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Cryo-EM structure of the inner ring from the Xenopus laevis nuclear pore complex. \u003cem\u003eCell Research\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 451-460 (2022). https://doi.org:10.1038/s41422-022-00633-x\u003c/li\u003e\n \u003cli\u003eZhang, Y.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Molecular architecture of the luminal ring of the Xenopus laevis nuclear pore complex. \u003cem\u003eCell research\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 532-540 (2020).\u003c/li\u003e\n \u003cli\u003eZhu, X.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Structure of the cytoplasmic ring of the Xenopus laevis nuclear pore complex. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e376\u003c/strong\u003e, eabl8280 (2022).\u003c/li\u003e\n \u003cli\u003eVon Appen, A.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e In situ structural analysis of the human nuclear pore complex. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e526\u003c/strong\u003e, 140-143 (2015).\u003c/li\u003e\n \u003cli\u003eSanchez Carrillo, I. B., Hoffmann, P. C., Barff, T., Beck, M. \u0026amp; Germain, H. Preparing Arabidopsis thaliana root protoplasts for cryo electron tomography. \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e (2023). https://doi.org:10.3389/fpls.2023.1261180\u003c/li\u003e\n \u003cli\u003eKoornneef, M. \u0026amp; Meinke, D. The development of Arabidopsis as a model plant. \u003cem\u003eThe Plant Journal\u003c/em\u003e \u003cstrong\u003e61\u003c/strong\u003e, 909-921 (2010). https://doi.org:https://doi.org/10.1111/j.1365-313X.2009.04086.x\u003c/li\u003e\n \u003cli\u003eKr\u0026auml;mer, U. Planting molecular functions in an ecological context with Arabidopsis thaliana. \u003cem\u003eeLife\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, e06100 (2015). https://doi.org:10.7554/eLife.06100\u003c/li\u003e\n \u003cli\u003eAnalysis of the genome sequence of the flowering plant Arabidopsis thaliana. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e408\u003c/strong\u003e, 796-815 (2000). https://doi.org:10.1038/35048692\u003c/li\u003e\n \u003cli\u003eWoodward, A. W. \u0026amp; Bartel, B. Biology in Bloom: A Primer on the Arabidopsis thaliana Model System. \u003cem\u003eGenetics\u003c/em\u003e \u003cstrong\u003e208\u003c/strong\u003e, 1337-1349 (2018). https://doi.org:10.1534/genetics.118.300755\u003c/li\u003e\n \u003cli\u003eDavey, M. R., Anthony, P., Power, J. B. \u0026amp; Lowe, K. C. Plant protoplasts: status and biotechnological perspectives. \u003cem\u003eBiotechnology Advances\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 131-171 (2005). https://doi.org:https://doi.org/10.1016/j.biotechadv.2004.09.008\u003c/li\u003e\n \u003cli\u003eReyna-Llorens, I., Ferro-Costa, M. \u0026amp; Burgess, S. J. Plant protoplasts in the age of synthetic biology. \u003cem\u003eJournal of Experimental Botany\u003c/em\u003e (2023). https://doi.org:10.1093/jxb/erad172\u003c/li\u003e\n \u003cli\u003eShaw, R., Tian, X. \u0026amp; Xu, J. Single-Cell Transcriptome Analysis in Plants: Advances and Challenges. \u003cem\u003eMolecular Plant\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 115-126 (2021). https://doi.org:10.1016/j.molp.2020.10.012\u003c/li\u003e\n \u003cli\u003eSheen, J. Signal transduction in maize and Arabidopsis mesophyll protoplasts. \u003cem\u003ePlant Physiol\u003c/em\u003e \u003cstrong\u003e127\u003c/strong\u003e, 1466-1475 (2001).\u003c/li\u003e\n \u003cli\u003eYoo, S.-D., Cho, Y.-H. \u0026amp; Sheen, J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. \u003cem\u003eNature Protocols\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 1565-1572 (2007). https://doi.org:10.1038/nprot.2007.199\u003c/li\u003e\n \u003cli\u003eSchaffer, M.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Optimized cryo-focused ion beam sample preparation aimed at in situ structural studies of membrane proteins. \u003cem\u003eJournal of Structural Biology\u003c/em\u003e \u003cstrong\u003e197\u003c/strong\u003e, 73-82 (2017). https://doi.org:https://doi.org/10.1016/j.jsb.2016.07.010\u003c/li\u003e\n \u003cli\u003eSaxena, P. K., Fowke, L. C. \u0026amp; King, J. An efficient procedure for isolation of nuclei from plant protoplasts. \u003cem\u003eProtoplasma\u003c/em\u003e \u003cstrong\u003e128\u003c/strong\u003e, 184-189 (1985). https://doi.org:10.1007/BF01276340\u003c/li\u003e\n \u003cli\u003eTamura, K., Fukao, Y., Iwamoto, M., Haraguchi, T. \u0026amp; Hara-Nishimura, I. Identification and Characterization of Nuclear Pore Complex Components in Arabidopsis thaliana. \u003cem\u003eThe Plant Cell\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 4084 (2010). https://doi.org:10.1105/tpc.110.079947\u003c/li\u003e\n \u003cli\u003eTamura, K. \u0026amp; Hara-Nishimura, I. The molecular architecture of the plant nuclear pore complex. \u003cem\u003eJournal of Experimental Botany\u003c/em\u003e \u003cstrong\u003e64\u003c/strong\u003e, 823-832 (2012). https://doi.org:10.1093/jxb/ers258\u003c/li\u003e\n \u003cli\u003eTang, Y., Huang, A. \u0026amp; Gu, Y. Global profiling of plant nuclear membrane proteome in Arabidopsis. \u003cem\u003eNature Plants\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 838-847 (2020). https://doi.org:10.1038/s41477-020-0700-9\u003c/li\u003e\n \u003cli\u003eHolzer, G. \u0026amp; Antonin, W. Breaking the Y. \u003cem\u003ePLOS Genetics\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, e1008109 (2019). https://doi.org:10.1371/journal.pgen.1008109\u003c/li\u003e\n \u003cli\u003eAsakawa, H.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Asymmetrical localization of Nup107-160 subcomplex components within the nuclear pore complex in fission yeast. \u003cem\u003ePLOS Genetics\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, e1008061 (2019). https://doi.org:10.1371/journal.pgen.1008061\u003c/li\u003e\n \u003cli\u003eBelgareh , N. m.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e An evolutionarily conserved NPC subcomplex, which redistributes in part to kinetochores in mammalian cells. \u003cem\u003eJournal of Cell Biology\u003c/em\u003e \u003cstrong\u003e154\u003c/strong\u003e, 1147-1160 (2001). https://doi.org:10.1083/jcb.200101081\u003c/li\u003e\n \u003cli\u003eBinder, A. \u0026amp; Parniske, M. Analysis of the Lotus japonicus nuclear pore NUP107-160 subcomplex reveals pronounced structural plasticity and functional redundancy. \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e (2014). https://doi.org:10.3389/fpls.2013.00552\u003c/li\u003e\n \u003cli\u003eKelley, K., Knockenhauer, K. E., Kabachinski, G. \u0026amp; Schwartz, T. U. Atomic structure of the Y complex of the nuclear pore. \u003cem\u003eNature Structural \u0026amp; Molecular Biology\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 425-431 (2015). https://doi.org:10.1038/nsmb.2998\u003c/li\u003e\n \u003cli\u003eLutzmann, M., Kunze, R., Buerer, A., Aebi, U. \u0026amp; Hurt, E. Modular self-assembly of a Y-shaped multiprotein complex from seven nucleoporins. \u003cem\u003eThe EMBO Journal\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 387-397 (2002). https://doi.org:https://doi.org/10.1093/emboj/21.3.387\u003c/li\u003e\n \u003cli\u003eStuwe, T.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Architecture of the nuclear pore complex coat. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e347\u003c/strong\u003e, 1148-1152 (2015). https://doi.org:doi:10.1126/science.aaa4136\u003c/li\u003e\n \u003cli\u003eWalther, T. C.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e The Conserved Nup107-160 Complex Is Critical for Nuclear Pore Complex Assembly. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e113\u003c/strong\u003e, 195-206 (2003). https://doi.org:10.1016/S0092-8674(03)00235-6\u003c/li\u003e\n \u003cli\u003eWiermer, M.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Putative members of the Arabidopsis Nup107-160 nuclear pore sub-complex contribute to pathogen defense. \u003cem\u003eThe Plant Journal\u003c/em\u003e \u003cstrong\u003e70\u003c/strong\u003e, 796-808 (2012). https://doi.org:https://doi.org/10.1111/j.1365-313X.2012.04928.x\u003c/li\u003e\n \u003cli\u003eBui, Khanh H.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Integrated Structural Analysis of the Human Nuclear Pore Complex Scaffold. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e155\u003c/strong\u003e, 1233-1243 (2013). https://doi.org:10.1016/j.cell.2013.10.055\u003c/li\u003e\n \u003cli\u003eJumper, J.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Highly accurate protein structure prediction with AlphaFold. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e596\u003c/strong\u003e, 583-589 (2021). https://doi.org:10.1038/s41586-021-03819-2\u003c/li\u003e\n \u003cli\u003eVaradi, M.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e AlphaFold Protein Structure Database in 2024: providing structure coverage for over 214 million protein sequences. \u003cem\u003eNucleic Acids Research\u003c/em\u003e \u003cstrong\u003e52\u003c/strong\u003e, D368-D375 (2023). https://doi.org:10.1093/nar/gkad1011\u003c/li\u003e\n \u003cli\u003eZhang, Y. \u0026amp; Skolnick, J. TM-align: a protein structure alignment algorithm based on the TM-score. \u003cem\u003eNucleic Acids Research\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 2302-2309 (2005). https://doi.org:10.1093/nar/gki524\u003c/li\u003e\n \u003cli\u003eFaure, G.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e iPBAvizu: a PyMOL plugin for an efficient 3D protein structure superimposition approach. \u003cem\u003eSource Code for Biology and Medicine\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 5 (2019). https://doi.org:10.1186/s13029-019-0075-3\u003c/li\u003e\n \u003cli\u003eVaradi, M.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. \u003cem\u003eNucleic Acids Research\u003c/em\u003e \u003cstrong\u003e50\u003c/strong\u003e, D439-D444 (2021). https://doi.org:10.1093/nar/gkab1061\u003c/li\u003e\n \u003cli\u003eMakarov, A. A., Padilla-Mejia, N. E. \u0026amp; Field, M. C. Evolution and diversification of the nuclear pore complex. \u003cem\u003eBiochemical Society Transactions\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, 1601-1619 (2021). https://doi.org:10.1042/bst20200570\u003c/li\u003e\n \u003cli\u003eTang, Y.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Proxiome assembly of the plant nuclear pore reveals an essential hub for gene expression regulation. \u003cem\u003eNature Plants\u003c/em\u003e (2024). https://doi.org:10.1038/s41477-024-01698-9\u003c/li\u003e\n \u003cli\u003eFernandez-Martinez, J. \u0026amp; Rout, M. P. One Ring to Rule them All? Structural and Functional Diversity in the Nuclear Pore Complex. \u003cem\u003eTrends in Biochemical Sciences\u003c/em\u003e \u003cstrong\u003e46\u003c/strong\u003e, 595-607 (2021). https://doi.org:10.1016/j.tibs.2021.01.003\u003c/li\u003e\n \u003cli\u003eHoffmann, P. C.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Nuclear pores as conduits for fluid flow during osmotic stress. \u003cem\u003ebioRxiv\u003c/em\u003e, 2024.2001.2017.575985 (2024). https://doi.org:10.1101/2024.01.17.575985\u003c/li\u003e\n \u003cli\u003eKlughammer, N.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Diameter Dependence of Transport through Nuclear Pore Complex Mimics Studied Using Optical Nanopores. (2023). https://doi.org:10.1101/2023.02.18.529008\u003c/li\u003e\n \u003cli\u003eGallem\u0026iacute;, M.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e DRACULA2 is a dynamic nucleoporin with a role in regulating the shade avoidance syndrome in Arabidopsis. \u003cem\u003eDevelopment\u003c/em\u003e \u003cstrong\u003e143\u003c/strong\u003e, 1623-1631 (2016).\u003c/li\u003e\n \u003cli\u003eIto, N.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Nuclear pore complex proteins are involved in centromere distribution. \u003cem\u003eIscience\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e (2024).\u003c/li\u003e\n \u003cli\u003eJiang, S.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Nucleoporin Nup98 participates in flowering regulation in a CONSTANS-independent mode. \u003cem\u003ePlant Cell Reports\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 1263-1271 (2019). https://doi.org:10.1007/s00299-019-02442-w\u003c/li\u003e\n \u003cli\u003eMermet, S.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Evolutionarily conserved protein motifs drive interactions between the plant nucleoskeleton and nuclear pores. \u003cem\u003eThe Plant Cell\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 4284-4303 (2023). https://doi.org:10.1093/plcell/koad236\u003c/li\u003e\n \u003cli\u003eNeumann, N., Jeffares, D. C. \u0026amp; Poole, A. M. Outsourcing the nucleus: nuclear pore complex genes are no longer encoded in nucleomorph genomes. \u003cem\u003eEvolutionary Bioinformatics\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 117693430600200023 (2006).\u003c/li\u003e\n \u003cli\u003eTeimer, R., Kosinski, J., von Appen, A., Beck, M. \u0026amp; Hurt, E. A short linear motif in scaffold Nup145C connects Y-complex with pre-assembled outer ring Nup82 complex. \u003cem\u003eNature Communications\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 1107 (2017). https://doi.org:10.1038/s41467-017-01160-9\u003c/li\u003e\n \u003cli\u003eDong, C.-H., Agarwal, M., Zhang, Y., Xie, Q. \u0026amp; Zhu, J.-K. The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e \u003cstrong\u003e103\u003c/strong\u003e, 8281-8286 (2006). https://doi.org:doi:10.1073/pnas.0602874103\u003c/li\u003e\n \u003cli\u003eHan, S.-H., Park, Y.-J. \u0026amp; Park, C.-M. Publisher Correction: HOS1 activates DNA repair systems to enhance plant thermotolerance. \u003cem\u003eNature Plants\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 237-237 (2021). https://doi.org:10.1038/s41477-020-00842-5\u003c/li\u003e\n \u003cli\u003eLee, K. \u0026amp; Seo, P. J. The Arabidopsis E3 ubiquitin ligase HOS1 contributes to auxin biosynthesis in the control of hypocotyl elongation. \u003cem\u003ePlant Growth Regulation\u003c/em\u003e \u003cstrong\u003e76\u003c/strong\u003e, 157-165 (2015). https://doi.org:10.1007/s10725-014-9985-x\u003c/li\u003e\n \u003cli\u003eMargalha, L.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e HOS1 promotes plant tolerance to low-energy stress via the SnRK1 protein kinase. \u003cem\u003eThe Plant Journal\u003c/em\u003e \u003cstrong\u003e115\u003c/strong\u003e, 627-641 (2023). https://doi.org:https://doi.org/10.1111/tpj.16250\u003c/li\u003e\n \u003cli\u003eShkryl, Y.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e CRISPR/Cas9-Mediated Knockout of HOS1 Reveals Its Role in the Regulation of Secondary Metabolism in Arabidopsis thaliana. \u003cem\u003ePlants\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 104 (2021).\u003c/li\u003e\n \u003cli\u003eMacGregor, D. R. \u0026amp; Penfield, S. Exploring the pleiotropy of hos1. \u003cem\u003eJournal of Experimental Botany\u003c/em\u003e \u003cstrong\u003e66\u003c/strong\u003e, 1661-1671 (2015). https://doi.org:10.1093/jxb/erv022\u003c/li\u003e\n \u003cli\u003eXiao, L.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Two Nucleoporin98 homologous genes jointly participate in the regulation of starch degradation to repress senescence in Arabidopsis. \u003cem\u003eBMC Plant Biology\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 292 (2020). https://doi.org:10.1186/s12870-020-02494-1\u003c/li\u003e\n \u003cli\u003eBeck, M., Lučić, V., F\u0026ouml;rster, F., Baumeister, W. \u0026amp; Medalia, O. Snapshots of nuclear pore complexes in action captured by cryo-electron tomography. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e449\u003c/strong\u003e, 611-615 (2007). https://doi.org:10.1038/nature06170\u003c/li\u003e\n \u003cli\u003eO\u0026rsquo;Malley, M. A., Leger, M. M., Wideman, J. G. \u0026amp; Ruiz-Trillo, I. Concepts of the last eukaryotic common ancestor. \u003cem\u003eNature Ecology \u0026amp; Evolution\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 338-344 (2019). https://doi.org:10.1038/s41559-019-0796-3\u003c/li\u003e\n \u003cli\u003eSchuller, A. P.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e The cellular environment shapes the nuclear pore complex architecture. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e598\u003c/strong\u003e, 667-671 (2021). https://doi.org:10.1038/s41586-021-03985-3\u003c/li\u003e\n \u003cli\u003eZila, V.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Cone-shaped HIV-1 capsids are transported through intact nuclear pores. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e184\u003c/strong\u003e, 1032-1046.e1018 (2021). https://doi.org:10.1016/j.cell.2021.01.025\u003c/li\u003e\n \u003cli\u003eBargmann, B. O. \u0026amp; Birnbaum, K. D. Fluorescence activated cell sorting of plant protoplasts. \u003cem\u003eJ Vis Exp\u003c/em\u003e (2010). https://doi.org:10.3791/1673\u003c/li\u003e\n \u003cli\u003eNesvizhskii, A. I., Keller, A., Kolker, E. \u0026amp; Aebersold, R. A statistical model for identifying proteins by tandem mass spectrometry. \u003cem\u003eAnalytical chemistry\u003c/em\u003e \u003cstrong\u003e75\u003c/strong\u003e, 4646-4658 (2003).\u003c/li\u003e\n \u003cli\u003eIshihama, Y.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Exponentially Modified Protein Abundance Index (emPAI) for Estimation of Absolute Protein Amount in Proteomics by the Number of Sequenced Peptides per Protein * S. \u003cem\u003eMolecular \u0026amp; Cellular Proteomics\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 1265-1272 (2005). https://doi.org:10.1074/mcp.M500061-MCP200\u003c/li\u003e\n \u003cli\u003eBeck, M., Mosalaganti, S. \u0026amp; Kosinski, J. From the resolution revolution to evolution: structural insights into the evolutionary relationships between vesicle coats and the nuclear pore. \u003cem\u003eCurrent Opinion in Structural Biology\u003c/em\u003e \u003cstrong\u003e52\u003c/strong\u003e, 32-40 (2018). https://doi.org:https://doi.org/10.1016/j.sbi.2018.07.012\u003c/li\u003e\n \u003cli\u003eWan, W.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Structure and assembly of the Ebola virus nucleocapsid. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e551\u003c/strong\u003e, 394-397 (2017). https://doi.org:10.1038/nature24490\u003c/li\u003e\n \u003cli\u003eMastronarde, D. N. \u0026amp; Held, S. R. Automated tilt series alignment and tomographic reconstruction in IMOD. \u003cem\u003eJournal of Structural Biology\u003c/em\u003e \u003cstrong\u003e197\u003c/strong\u003e, 102-113 (2017). https://doi.org:https://doi.org/10.1016/j.jsb.2016.07.011\u003c/li\u003e\n \u003cli\u003eHoffmann, P. C.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Structures of the eukaryotic ribosome and its translational states in situ. \u003cem\u003eNature Communications\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 7435 (2022). https://doi.org:10.1038/s41467-022-34997-w\u003c/li\u003e\n \u003cli\u003eTuroňov\u0026aacute;, B., Schur, F. K. M., Wan, W. \u0026amp; Briggs, J. A. G. Efficient 3D-CTF correction for cryo-electron tomography using NovaCTF improves subtomogram averaging resolution to 3.4\u0026Aring;. \u003cem\u003eJournal of Structural Biology\u003c/em\u003e \u003cstrong\u003e199\u003c/strong\u003e, 187-195 (2017). https://doi.org:https://doi.org/10.1016/j.jsb.2017.07.007\u003c/li\u003e\n \u003cli\u003eAllegretti, M.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e In-cell architecture of the nuclear pore and snapshots of its turnover. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e586\u003c/strong\u003e, 796-800 (2020). https://doi.org:10.1038/s41586-020-2670-5\u003c/li\u003e\n \u003cli\u003eErmel, U. H., Arghittu, S. M. \u0026amp; Frangakis, A. S. ArtiaX: An electron tomography toolbox for the interactive handling of sub-tomograms in UCSF ChimeraX. \u003cem\u003eProtein Science\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, e4472 (2022). https://doi.org:https://doi.org/10.1002/pro.4472\u003c/li\u003e\n \u003cli\u003eGoddard, T. D.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e UCSF ChimeraX: Meeting modern challenges in visualization and analysis. \u003cem\u003eProtein Science\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 14-25 (2018). https://doi.org:https://doi.org/10.1002/pro.3235\u003c/li\u003e\n \u003cli\u003eEvans, R.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Protein complex prediction with AlphaFold-Multimer. \u003cem\u003ebioRxiv\u003c/em\u003e, 2021.2010.2004.463034 (2022). https://doi.org:10.1101/2021.10.04.463034\u003c/li\u003e\n \u003cli\u003eYu, D., Chojnowski, G., Rosenthal, M. \u0026amp; Kosinski, J. AlphaPulldown\u0026mdash;a python package for protein\u0026ndash;protein interaction screens using AlphaFold-Multimer. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e39\u003c/strong\u003e (2022). https://doi.org:10.1093/bioinformatics/btac749\u003c/li\u003e\n \u003cli\u003eRantos, V., Karius, K. \u0026amp; Kosinski, J. Integrative structural modeling of macromolecular complexes using Assembline. \u003cem\u003eNature Protocols\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 152-176 (2022). https://doi.org:10.1038/s41596-021-00640-z\u003c/li\u003e\n \u003cli\u003eWebb, B.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Integrative structure modeling with the Integrative Modeling Platform. \u003cem\u003eProtein Science\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 245-258 (2018). https://doi.org:https://doi.org/10.1002/pro.3311\u003c/li\u003e\n \u003cli\u003eSaltzberg, D.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e in \u003cem\u003eBiomolecular Simulations: Methods and Protocols\u003c/em\u003e (eds Massimiliano Bonomi \u0026amp; Carlo Camilloni) 353-377 (Springer New York, 2019).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Higher plants, subtomogram averaging, cryo-ET, cryo-FIB, nucleoporin","lastPublishedDoi":"10.21203/rs.3.rs-6197803/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6197803/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The nucleus is delimited by the nuclear envelope (NE), where nuclear pore complexes (NPCs) are embedded. While this complex has been well studied in vertebrates, yeast, and, more recently, in algae, in situ structural data of higher plants is still missing. Here, we show that many individual nucleoporins of Arabidopsis thaliana and human present high structural similarity. We report a first higher plant in situ NPC structure, derived from A. thaliana root protoplasts using cryo electron tomography, subtomogram averaging and homology-based integrative modeling. We present a plant NPC model based on predicted models of A. thaliana NUPs identified by mass spectrometry. The plant NPC scaffold exhibits differences in diameter and height to C. reinhardtii, while sharing some structural features with H. sapiens NPCs. Notably, we observed that the A. thaliana NPC contains NUP155 connectors like the H. sapiens NPC, which may explain the height difference when compared to the C. reinhardtii NPC.","manuscriptTitle":"In situ architecture of the nuclear pore complex of the higher plant Arabidopsis thaliana","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-24 03:57:31","doi":"10.21203/rs.3.rs-6197803/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-plants","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nplants","sideBox":"Learn more about [Nature Plants](http://www.nature.com/nplants/)","snPcode":"","submissionUrl":"","title":"Nature Plants","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"cff31d09-4477-4b82-8d23-3e77475a7d6e","owner":[],"postedDate":"March 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":45912068,"name":"Biological sciences/Cell biology/Cell signalling/Nuclear receptors"},{"id":45912069,"name":"Biological sciences/Plant sciences/Plant cell biology"}],"tags":[],"updatedAt":"2025-11-01T07:10:57+00:00","versionOfRecord":{"articleIdentity":"rs-6197803","link":"https://doi.org/10.1038/s41477-025-02138-y","journal":{"identity":"nature-plants","isVorOnly":false,"title":"Nature Plants"},"publishedOn":"2025-10-31 04:00:00","publishedOnDateReadable":"October 31st, 2025"},"versionCreatedAt":"2025-03-24 03:57:31","video":"","vorDoi":"10.1038/s41477-025-02138-y","vorDoiUrl":"https://doi.org/10.1038/s41477-025-02138-y","workflowStages":[]},"version":"v1","identity":"rs-6197803","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6197803","identity":"rs-6197803","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}