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Integrative structure and functional anatomy of a nuclear pore complex

Nature volume 555, pages 475–482 (2018)Cite this article

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Abstract

Nuclear pore complexes play central roles as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm. However, their large size and dynamic nature have impeded a full structural and functional elucidation. Here we determined the structure of the entire 552-protein nuclear pore complex of the yeast Saccharomyces cerevisiae at sub-nanometre precision by satisfying a wide range of data relating to the molecular arrangement of its constituents. The nuclear pore complex incorporates sturdy diagonal columns and connector cables attached to these columns, imbuing the structure with strength and flexibility. These cables also tie together all other elements of the nuclear pore complex, including membrane-interacting regions, outer rings and RNA-processing platforms. Inwardly directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized into distinct functional units. This integrative structure enables us to rationalize the architecture, transport mechanism and evolutionary origins of the nuclear pore complex.

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Figure 1: Defining the mass, composition and stoichiometry of the native NPC.
Figure 2: Chemical cross-linking and mass spectrometry reveals nucleoporin connectivity in the NPC.
Figure 3: Morphology of the NPC.
Figure 4: Structural dissection of the NPC.
Figure 5: Key NPC architectural features and principles.
Figure 6: The distribution of FG repeats informs the NPC transport gating mechanism.

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Change history

  • 21 March 2018

    Minor changes were made to the descriptions of the Supplementary Information files.

  • 06 April 2018

    The links for the three PDB-dev accessions were added to the HTML.

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Acknowledgements

We thank B. Webb (UCSF) for help with the Integrative Modelling Platform, the Rockefeller University Outreach Program for support for A.S.C., the NYULMC OCS Microscopy Core, K. Uryu and the EMRC Resource Center (Rockefeller University) for assistance with negative-stain electron microscopy, F. Alber, M. C. Field, N. Ketaren, S. Obado, R. Hayama and D. Simon for feedback and critical reading of the manuscript, and L. Herlands for support and encouragement. The work was supported by a NSF GRF 1650113 (I.E.C.), a NSF grant CHE-1531823 (M.F.J.), the SIMR (J.L.G.), NIH grants R01 GM080477 (J.L.G.), U54 GM103511 (B.T.C., A.S., J.D.A. and M.P.R.), R01 GM112108 (M.P.R. and J.D.A.), P41 GM109824 (M.P.R., A.S., J.D.A. and B.T.C.), P50 GM076547 (J.D.A.), R01 GM063834 (C.W.A.), R01 GM080139 (S.J.L.), P41 GM103314 (B.T.C.), R01 GM083960 (A.S.) and U54 DK107981 (M.P.R. and J.D.A.). We are grateful for the support provided by G. Blobel, who inspired the work presented here.

Author information

Author notes

  1. Riccardo Pellarin

    Present address: Structural Bioinformatics Unit, Institut Pasteur, CNRS UMR 3528, Paris, France

  2. Yi Shi

    Present address: Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, 15260, USA

  3. M. Jason de la Cruz

    Present address: Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, New York, 10065, USA

  4. Seung Joong Kim, Javier Fernandez-Martinez, Ilona Nudelman, Yi Shi and Wenzhu Zhang: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Bioengineering and Therapeutic Sciences Department of Pharmaceutical Chemistry, and California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, 94158, California, USA

    Seung Joong Kim, Barak Raveh, Ilan E. Chemmama, Riccardo Pellarin, Ignacia Echeverria, Charles H. Greenberg & Andrej Sali

  2. Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, 10065, New York, USA

    Javier Fernandez-Martinez, Ilona Nudelman, Azraa S. Chaudhury, Rosemary Williams, Roxana Mironska & Michael P. Rout

  3. Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, 10065, New York, USA

    Yi Shi, Wenzhu Zhang, Junjie Wang, Erica Y. Jacobs & Brian T. Chait

  4. Institute for Systems Biology, 401 Terry Ave, N., Seattle, 98109, Washington, USA

    Thurston Herricks & John D. Aitchison

  5. Stowers Institute for Medical Research, Kansas City, 64110, Missouri, USA

    Brian D. Slaughter, Manjunatha Shivaraju, Jay R. Unruh & Jennifer L. Gerton

  6. Department of Chemistry, Indiana University, Bloomington, Indiana, 47405, USA

    Joanna A. Hogan & Martin F. Jarrold

  7. Skirball Institute and Department of Cell Biology, New York University School of Medicine, New York, 10016, New York, USA

    Paula Upla & David L. Stokes

  8. Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, 20148, Virginia, USA

    Zhiheng Yu & M. Jason de la Cruz

  9. Center for Infectious Disease Research, Seattle, 98109, Washington, USA

    John D. Aitchison

  10. Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, 77030, Texas, USA

    Steven J. Ludtke

  11. Department of Physiology and Biophysics, Boston University School of Medicine, 700 Albany Street, Boston, 02118, Massachusetts, USA

    Christopher W. Akey

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Contributions

The order of first co-authors was determined through a random selection process. I.N., J.F.-M., A.S.C., R.W. and M.P.R. performed the affinity purifications; W.Z., J.F.-M., R.W., R.M., E.Y.J., M.P.R. and B.T.C. performed the quantitative mass spectrometry; M.S., B.D.S., J.R.U. and J.L.G. performed the calibrated imaging; J.A.H.. B.T.C. and M.F.J. performed the charge detection mass spectrometry; Y.S., J.F.-M., R.W., I.N., J.W. and B.T.C. performed the chemical crosslinking with mass spectrometry; C.W.A., S.J.L., I.N., Z.Y. and M.J.d.l.C. performed the cryo-ET; S.J.K. performed the small-angle X-ray scattering; T.H., J.F.-M. and J.D.A. performed the phenotypic profiling; P.U. and D.L.S. performed the negative-stain electron microscopy; S.J.K., B.R., I.E.C., R.P., I.E., C.H.G. and A.S. performed the integrative structure computations; S.J.L., C.W.A., B.T.C., A.S. and M.P.R. supervised the project; S.J.K., J.F.-M., I.N., Y.S., W.Z., B.R., S.J.L., C.W.A., B.T.C., A.S. and M.P.R. wrote the manuscript.

Corresponding authors

Correspondence to Steven J. Ludtke, Christopher W. Akey, Brian T. Chait, Andrej Sali or Michael P. Rout.

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Extended data figures and tables

Extended Data Figure 1 Integrative structure determination of the S. cerevisiae NPC at 9 Å precision.

a, Schematic of integrative structure determination of the S. cerevisiae NPC. Random initial structures of the Nups and their sub-complexes were optimized by satisfying spatial restraints implied by the input information. b, The full description of integrative structure determination of the S. cerevisiae NPC, proceeded through four stages8,90,91,92 (Supplementary Table 3): (1) gathering data, (2) representing subunits and translating data into spatial restraints, (3) configurational sampling to produce an ensemble of structures that satisfies the restraints and (4) analysing and validating the ensemble structures and data (Extended Data Figs 7, 8Supplementary Tables 2–4 and Methods). The integrative structure modelling protocol (stages 2, 3 and 4) was scripted using the Python modelling interface package version 4d97507, which is a library for modelling macromolecular complexes based on our open-source IMP package90 version 2.6 (https://integrativemodeling.org). c, Convergence of the structure score for the 5,529 good-scoring NPC structures; the scores do not continue to improve as more structures are computed, essentially independently of each other. The error bar represents the standard deviations of the best scores, estimated by repeating sampling of NPC structures ten times (n?=?10, mean score values plotted). The red dotted line indicates the total score threshold (88,644.1) that defines the good-scoring NPC structures (Methods). d, Distribution of scores for structure samples 1 (red) and 2 (blue), comprising the 5,529 good-scoring NPC structures (nsample1?=?2,359 and nsample2?=?3,170 structures). The non-parametric Kolmogorov–Smirnov two-sample test120,121 (two-sided) indicates that the difference between the two score distributions is insignificant (P value (1.0)?>?0.05). In addition, the magnitude of the difference is small, as demonstrated by the Kolmogorov–Smirnov two-sample test statistic, D, of 0.045. Thus, the two score distributions are effectively equal. e, Three criteria for determining the sampling precision (y axis), evaluated as a function of the r.m.s.d. clustering threshold123 (x axis) (n?=?5,529 structures). First, the P value is computed using the χ2-test (one-sided) for homogeneity of proportions122 (red dots). Second, an effect size for the χ2-test is quantified by the Cramer’s V value (blue squares). Third, the population of structures in sufficiently large clusters (containing at least ten structures from each sample) is shown as green triangles. The vertical dotted grey line indicates the r.m.s.d. clustering threshold at which three conditions are satisfied (χ2-test P value (0.75)?>?0.05 (red, horizontal dotted line), Cramer’s V (0.065)?<?0.10 (blue, horizontal dotted line) and the population of clustered structures (0.90)?>?0.80 (green, horizontal dotted line)), thus defining the sampling precision of 9?Å. The three solid curves (in red, blue and green) were drawn through the points to help visualize the results. f, Population of sample 1 and 2 structures in the three clusters obtained by threshold-based clustering123 using an r.m.s.d. threshold of 12?Å. The dominant cluster (cluster 1) contains 80.3% of the structures. Cluster precision is shown for each cluster. The precision of the dominant cluster defines the structure precision.

Extended Data Figure 2 Quantitative analysis of the mass and stoichiometry of the endogenous NPC (part 1).

a, A multipronged approach to accurately define the mass, stoichiometry and composition of native macromolecular assemblies. Schematic is shown of the multiple orthologous methods that are integrated within our strategy for the analysis of native assemblies. The main experimental methods are listed on top, followed by the characteristic that they help to quantify (in blue) and the type of sample to which they were applied. The final outcome of each method is indicated (black arrows); the steps of each method that are compared to serve as a cross-check control are indicated (blue dashed lines). At bottom, the integration of the different data points into a final comprehensive description of the endogenous assembly is depicted. Small cartoon insets of the NPC are used to illustrate the analysis. b, SDS-PAGE analysis of the affinity-captured S. cerevisiae NPCs isolated from an MLP1–PPX-PrA tagged strain (n?>?20 independent experiments). Molecular weight marker values (Mw) are indicated to the left of the gel lane. Dots signify the main protein components of the isolated NPCs as identified by liquid chromatography–mass spectrometry (Extended Data Fig. 3c). Proteins are grouped and coloured by functional categories or membership of discrete macromolecular assemblies. Nups, blue; mRNA transport factors (TFs), red; transport factors, orange; transcription-export (TREX), green; contaminants and/or others, grey. For gel source data, see Supplementary Fig. 1. c, Cryo-electron microscopy analysis of the affinity-captured NPCs (n?>?20 independent experiments). The particles have a clear preferred orientation (Methods). Some side views are presented in the inset. The central transporter is present in every NPC (indicated by ‘T’). Scale bar, 1,000?Å. d, Schematic showing the primary amino acid sequence of the 148.2?kDa synthetic QconCAT-A. It includes two peptides for each Nup (thick bars), arranged in the indicated order. The native three amino acid residues flanking regions (thin bars) of each peptide were included to preserve the native trypsin target sequence. A N-terminal 3×FLAG tag was included, as well as a C-terminal 6×His tag for purification under denaturing conditions. The stringent criteria used for the selection of the QconCAT peptides are described in the Methods. e, MALDI mass spectrometry spectrum of intact, purified full-length QconCAT-A labelled with stable isotope, showing that a single species was detected. Numbers above peaks denote the QconCAT-A protein species with n positive charges and the 2M QconCAT-A protein dimer. The measured molecular weight of the QconCAT-A labelled with stable isotope was 149,049 ± 38 Da, consistent with its calculated molecular weight of 148,200 Da (Methods).

Extended Data Figure 3 Quantitative analysis of the mass and stoichiometry of the endogenous NPC (part 2).

a, Left, schematic localization of the Nup–GFP reporters selected for the in vivo calibrated imaging stoichiometry analyses. Nups were selected to represent every major NPC module and to provide comprehensive coverage of the assembly. Right, Kernel density estimation of distributions of GFP proteins per Nup were calculated from the calibrated imaging data. n?=?48–178. b, Heat map of a yeast cell expressing Nup85–GFP. Image (left) was acquired as described in Methods. In addition, for illustration purposes, a maximum projection along the z axis was performed, and the image was smoothed with a Gaussian blur of radius 1. A heat map was used to illustrate intensity units in raw photon counts. For the area outlined in a red rectangle, a 2D distribution of photon counts and the corresponding Gaussian fit are shown (right). c, Stoichiometries of main components associated with the affinity-captured NPCs, as determined by label-free mass spectrometry quantification (at least three peptides per protein). Proteins are grouped and coloured by functional categories or membership of discrete macromolecular assemblies. The TREX complex components are included in the ‘Transport & Associated Factors’ category and labelled with an asterisk. QconCAT-derived stoichiometries for all the Nups (dark grey bars) are shown for comparison.

Extended Data Figure 4 Cryo-ET strategy and the resulting 3D cryo-ET map of the NPC exhibiting non-enforced local C2-symmetry axes in the inner ring.

a, Diagram describing the methodology used to obtain the whole S. cerevisiae NPC cryo-ET map (Methods). b, 2D class averages are shown (protein in white), which were calculated using the original unaligned sub-tomograms projected along the z axis. The overall thickness of the S. cerevisiae NPC is apparent in a side-view class, and the local C2-symmetry axes in the inner ring are also apparent (indicated with ‘2’). The transporter density is present in every class. c, Left, top view of the cryo-ET map with the two local C2-symmetry axes indicated by arrows and labels (sym 1 and sym 2). They are 22.5° apart, owing to the C8-symmetry axis. Right, 2D projections of the top view, and two side views along the two local C2-symmetry axes (side 1 and side 2 projected along axes sym 1 and sym 2, respectively). d, Seven cross-sections of the cryo-ET map are shown on the right (labelled 1–7) with their positions in the 3D map indicated in the side view on the left. The local C2-symmetry of the inner ring is apparent in cross-sections 2–6, mirrored about the central section in panel 4. Labels throughout: C, cytoplasm; N, nucleoplasm; T, central transporter; S, core scaffold; MR, membrane ring; IR, inner ring. Scale bar, 100?Å.

Extended Data Figure 5 Resolution estimates for the cryo-ET map of the NPC and comparison of cross-sections between the intermediate, final and RELION cryo-ET maps.

a, b, Top (a) and side (b) views of the cryo-ET map are colour-coded according to local resolution estimates (colour bar), and are shown at a low threshold to reveal weaker density features at the periphery of the NPC that are more flexible. c, Cross-sections are shown at a reduced scale, colour coded according to local resolution estimates (colour bar). A remnant of the pore membrane (M) is present, encircling the entire mid-line of the NPC. Sections 3–5 are shown at a higher threshold. In section 3, the inner-ring region is indicated by ‘spokes’. In section 4, local C2-symmetry axes are indicated by dashed arrows. d, Thick sections of the inner ring are shown at a higher threshold, as viewed along the membrane plane. Note that the inner ring (indicated) is almost entirely in the 20–25?Å resolution range. C, cytoplasmic side; N, nuclear side; CR, cytoplasmic ring; NR, nuclear ring; M, pore membrane; T, central transporter. e, Comparison of five cross-sections (cross-section number on left) in the inner-ring region of the NPC between cryo-ET maps in different stages of the reconstruction process (Extended Data Fig. 4a): intermediate map (left column), final map (middle column) and an independent validation map, reconstructed with RELION at a twice-reduced Å per pixel size (right column). Details on the reconstruction of maps are provided in Methods.

Extended Data Figure 6 2D classification of projections from 1,864 original NPC sub-tomograms aligned with their C8-symmetry axis nearly along the z axis.

a, In total, 18 good class averages are shown after maximum likelihood classification (using RELION 1.481,82) without symmetry imposed. Each class average (on the left) is paired with a C8-symmetry enforced image of itself (on the right). Central transporter densities are present in each of the class averages (both with and without imposed C8-symmetry), indicating that the central transporter is generally present in these particles. b, An expanded view of a large class from a shows bridges (indicated) between the core scaffold and the central transporter, both before (left) and after averaging (right) using the C8-symmetry of the NPC. S, core scaffold; MR, membrane ring; T, central transporter. Matching panels in a and b are marked with white dots. c, d, The cryo-ET 3D map is presented as in Fig. 3 and is zoomed in to show the meshwork of bridges between the scaffold and the central transporter, as viewed from the cytoplasm and the nucleoplasm, respectively.

Extended Data Figure 7 Validation of the NPC structure (part 1).

a–c, Satisfaction of the chemical cross-links. a, Identified chemical cross-links were mapped onto the integrative structure of the entire NPC, as shown in front (upper) and top (lower) views. Satisfied cross-links, with Cα–Cα distances that fall within the distance threshold of 35?Å in at least one good-scoring NPC structure, are shown in blue. Violated cross-links, with Cα–Cα distances that are larger than 35?Å, are shown in orange. The histogram on the right shows the distribution of the cross-linked Cα–Cα distances, validating the NPC structure. b, Mapping of the cross-links onto the cytoplasmic and nucleoplasmic connector Nups (Nup116, Nup100, Nup145N, Nup1 and Nup60). Front (right) and side (left) views show how the NPC outer rings are connected to the inner ring through a network of connector Nups across the length of the spoke. c, Mapping of the cross-links onto the inner (and membrane) and outer rings, in front (upper) and top (lower) views. d–g, Satisfaction of data and considerations that were not used to compute the structure. d, Our integrative structure of the NPC (left) was compared a previously published topological map3,8 (right). The two structures are consistent with each other, though our integrative structure is defined at an order of magnitude higher precision. e, Satisfaction of affinity purification and overlay assays data (composites); our current structure satisfies all 82 composites determined by affinity purification and overlay assays3,8, even though these data were not used in its determination. For example, Pom152, Pom34, Ndc1, Nup157 and Nup170 are connected with each other (left), consistent with the previously published composites determined using the affinity purification data3,8 (right). f, Satisfaction of SAXS data; the atomic structures of eight Nups are consistent with the corresponding SAXS profiles for their constructs9,12,23,83,84,85,86 (Supplementary Tables 2, 6 and Methods). For example, the SAXS profile calculated from the atomic structure of Pom152718–1148 (red curve) using FoXS111 is well-matched (χ?=?1.48) to the corresponding experimental SAXS profile23 (black dots; n?=?20 exposures). For visualization purposes, the Pom152718–1148 structure (represented as a ribbon) is shown along with the best fit of the ab initio shape (represented as a transparent envelope) computed from the experimental SAXS profile. g, Satisfaction of the negative-stain electron microscopy 2D class averages for the Nic96 complex; the structures of the Nic96 complex (composed of Nic96, Nsp1, Nup49 and Nup57) in the dominant cluster can be projected well on 2D class averages obtained for the natively isolated complex (n?=?5,458 particles; Methods). The experimental class averages were satisfied by the structure with cross-correlation coefficients of 0.85 and 0.80, respectively (Methods).

Extended Data Figure 8 Validation of the NPC structure (part 2), showing consistency between the NPC structure and the cryo-ET density map.

The cryo-ET density map is shown at a high-density threshold (grey) to reveal details of the inner ring. A representative structure of the inner ring is shown docked into the density, showing the excellent fit. All Nups are coloured as in Fig. 4. The pore membrane is indicated by M. a, Full eight-spoke inner ring (scale bar, 100 Å). b–d, front (b), top (c) and back (d) views of three spokes with neighbours coloured brown and grey (scale bar, 50 Å). e, Different views of a single spoke (scale bar, 50 Å) are shown within the density map. f, Thick cross-sections are shown through a single spoke in the inner ring, as viewed from the central C8-symmetry axis (scale bar, 50?Å); MBMs (see Fig. 5) are indicated.

Extended Data Figure 9 Functional analysis of the fitness of nucleoporin mutants using ODELAY.

a, The fitness defect phenotype was quantified and plotted (mean Z-score; n?=?6 experiments, containing at least 200–300 individuals per point; see Methods for details) for each nucleoporin truncation or C-terminal protein-A tagged mutant in order of decreasing fitness (increasing number of units), as observed by ODELAY assay14 (Methods). Strains for which truncations in a haploid background were found to lead to lethality after tetrad dissection (Nic96 and Nup192) were assigned the maximum level of defect and plotted on top of the rest (diploid), on the basis of the fitness phenotype observed for the indicated Nic96 and Nup192 mutants in a diploid background (in which a wild-type copy of the nucleoporin is also present and expressed). Six divisions were assigned based on decreasing levels of fitness9,13; white (wild type) to dark purple (severe defect). AU, arbitrary unit; error bar?=?standard deviation. b, Mapping of the colour code described in a into the NPC components. Horizontal lines represent the amino acid residue length of each protein and truncated version; amino acid residue positions are shown on top of the lines.

Extended Data Figure 10 Proposed evolutionary origin of the NPC from a later amalgam of membrane coating complexes.

a, Diagram depicting how the NPC may have originated from an ancestral coatomer module through a series of duplications, divergence and secondary loss events. Top, the origin of an ancestral proto-NPC coatomer module from an amalgamation of COPI-like and COPII-like complexes. Middle, the initial duplication leading to the origin of the inner and outer rings, and their associated coiled bundles. Presumed secondary losses removed the additional COPII-like subunit of the inner-ring protomer; loss of the adaptin-like subunit from the outer ring may have occurred here, or later in only certain lineages. Bottom, another duplication and divergence within each spoke may then have generated two parallel and laterally-offset paralogous columns; in the outer ring, a COPII-like subunit was then lost from one of the duplicates. The coiled bundles of the outer rings gave rise to the cytoplasmic export complex and nuclear basket by subsequent duplication; the export complex itself is a duplicate with a dimer of trimeric coiled bundles in its core. Outer-ring duplications are not shown. Relevant nucleoporin domains are depicted as follows: β-propellers (cyan circles), α-solenoids (pink bars) and coiled-coil domains (orange sticks). Left, diagrams (grey) exemplify the path of duplications within the whole NPC. Examples of ribbon representations for each module are presented. The anchoring points of the coiled-coil cytoplasmic Nup82 complex and the nuclear basket (orange densities) into an equivalent region of the outer-ring Nup84 complex (grey density) are shown. b, Conserved structural motifs connecting spokes in the outer and inner rings. Diagram showing how the spoke-to-spoke connection is established through similar head-to-head connections of heterodimers containing one COPI-like and one COPII-like subunits in both the outer (left) and the inner (right) NPC rings. Top, nucleoporin domains coloured as in a; bottom, COPI-like Nups in red, COPII-like Nups in blue.

Extended Data Figure 11 Position of the FG-repeat anchor points and heat mapping of the FG repeats.

a, Three views of the complete structure of the NPC are shown with major structural features (coloured as in Fig. 4 and Supplementary Table 2) and a snapshot of modelled FG-repeat regions (indicated in green). For each Nup, the localization probability density of the ensemble of structures is shown with a representative structure from the ensemble embedded within it. See also Supplementary Videos 1, 2, 3. Scale bar, 200?Å. b, Positions of FG-repeat anchor points within the ensemble of solutions are depicted as green surfaces; the Nups to which they belong are labelled in the centre image. Left, side view of three spokes; centre, side view of one spoke; right, top view of three spokes. Scale bar, 100?Å. c, Heat mapping of the type of FG-repeat region of each FG Nup (FXFG/FG type, red; GLFG type, blue), showing partitioning of the FG types to different regions of the central transporter. Identity of mapped Nups is shown in the diagram on the right. Scale bar.100?Å. d, Heat mapping of the effect on NPC permeability of the truncation of an FG repeat in each FG Nup, relative to the wild-type strain (p/pWT); the severity of the permeability defect is indicated in increasing intensity of shades of blue from minor defect (light green) to severe defect (dark blue), thereby defining the FG repeats that are most important in maintaining the passive permeability barrier. Identities of mapped Nups are shown in the diagram on the right. Scale bar, 100?Å.

Extended Data Figure 12 Comparison of the S. cerevisiae NPC structure and human NPC core scaffold.

a, Comparison between the inner rings in the structure of the S. cerevisiae NPC (first row) and the core scaffold of the human NPC (second row) (Protein Data Bank code: 5IJN6). Yeast Nups are coloured as in Fig. 4; human Nup homologues are coloured as their yeast counterparts. All copies of human Nup155 are coloured as yeast Nup157, and all copies of human Nup205/Nup188 are coloured as yeast Nup192. Only homologue Nups present in both yeast and human are shown. The human NPC core scaffold includes two additional copies of Nup155 that are absent in yeast (owing to the different stoichiometry between organisms). Yeast Nup53 and Nup59 are not shown because their counterparts are not present in the human NPC core scaffold. b, Major differences in the inner ring between the S. cerevisiae and human NPCs are highlighted, in the cross-sectional view near the equator. c, Positions of yeast Nups homologous to oncogenic human Nups (in parentheses) are shown in red, mapped onto three spokes of the NPC.

Supplementary information

Life Sciences Reporting Summary (PDF 72 kb)

Supplementary Information

This file contains the images from the Cryo-ET raw data (tilt series) and reconstructed tomograms, as well as the “Gold standard” refinement procedure for the final Cryo-ET map; and Supplementary Figure 1: Gel source data for Extended Data Fig. 2B. (PDF 11282 kb)

Supplementary Information

This file contains full supplementary table legends for tables 1-9, supplementary results and discussion, full supplementary video legends, and supplementary references. (PDF 484 kb)

Supplementary Tables

This file contains supplementary tables 1 -9. Supplementary Table 1 contains a list of 3,077 chemical cross-linked peptides identified via mass spectrometry. Supplementary Table 2 contains a representations of the S. cerevisiae NPC components (all 32 Nups) for integrative structure determination. Supplementary Table 3 contains a summary of the integrative structure determination, thoroughness of configurational sampling, and structure precision. Supplementary Table 4 contains a summary of spatial restraints used for the integrative structure determination and data satisfaction. Supplementary Table 5 contains a S. cerevisiae strains used in this study. Supplementary Table 6 contains an analysis of 147 SAXS profiles for 18 Nups. Supplementary Table 7 contains a list of peptides selected to construct QconCAT-A and B. Supplementary Table 8 contains a label-free MS analysis of native affinity captured NPCs and associated proteins. Supplementary Table 9 contains a cryo-electron tomographic data collection, processing, and refinement. (see Supplementary Information document for full table legends) (ZIP 4029 kb)

Supplementary Data

This file contains Source Data (part 1) for SAXS. It contains the SAXS data for Nup133, Nup120, Nup84, Nup85, Pom152, Nup145, Nup100, and Nup116. (ZIP 14369 kb)

Supplementary Data

This file contains Source Data (part 2) for SAXS. It contains the SAXS data for Nup188, Nup192, Nup2, Nup53, Nup59, Nup60, Nup82, Nsp1, Mlp1, and Mlp2. (ZIP 16649 kb)

Integrative structure and functional anatomy of the yeast Nuclear Pore Complex.

For each Nup (color-coded according to Supplementary Table 2B-H; same as Fig. 4), the localization probability density of the ensemble of structures is shown along with a representative atomic structure (where available) embedded within the localization density. A model of the pore membrane region is shown in light grey. First, we show a summary of the data used to compute the NPC structure. Second, the structure is rotated and shown in different orientations. (MP4 27862 kb)

Structural dissection of the yeast Nuclear Pore Complex.

Structural dissection of the yeast Nuclear Pore Complex. Using the same representation as in Supplementary Video 1, the structure of the NPC is dissected into its component modules and nucleoporins, emphasizing the spoke-to-spoke connections. (MP4 29186 kb)

Architectural and functional features of the yeast Nuclear Pore Complex.

Using the same representation as in Supplementary Video 1, first we show the position of the membrane interacting region, extended disordered connectors, and FG repeat anchor points are shown. Second, we a show a Brownian dynamics simulation of FG repeats. Third, we show the localization density of the FG repeats in the central channel. (MP4 19662 kb)

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Kim, S., Fernandez-Martinez, J., Nudelman, I. et al. Integrative structure and functional anatomy of a nuclear pore complex. Nature 555, 475–482 (2018). https://doi.org/10.1038/nature26003

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