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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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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

    • Yi Shi
    • , Riccardo Pellarin
    •  & M. Jason de la Cruz

    Present addresses: Structural Bioinformatics Unit, Institut Pasteur, CNRS UMR 3528, Paris, France (R.P.); Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260, USA (Y.S.); Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA (M.J.d.l.C.).

    • Seung Joong Kim
    • , Javier Fernandez-Martinez
    • , Ilona Nudelman
    • , Yi Shi
    •  & Wenzhu Zhang

    These authors contributed equally to this work.

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, California 94158, 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, New York 10065, 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, New York 10065, USA

    • Yi Shi
    • , Wenzhu Zhang
    • , Junjie Wang
    • , Erica Y. Jacobs
    •  & Brian T. Chait
  4. Institute for Systems Biology, 401 Terry Ave. N., Seattle, Washington 98109, USA

    • Thurston Herricks
    •  & John D. Aitchison
  5. Stowers Institute for Medical Research, Kansas City, Missouri 64110, 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, New York 10016, USA

    • Paula Upla
    •  & David L. Stokes
  8. Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, Virginia 20148, USA

    • Zhiheng Yu
    •  & M. Jason de la Cruz
  9. Center for Infectious Disease Research, Seattle, Washington 98109, USA

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

    • Steven J. Ludtke
  11. Department of Physiology and Biophysics, Boston University School of Medicine, 700 Albany Street, Boston, Massachusetts 02118, 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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

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

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Life Sciences Reporting Summary

  2. 2.

    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.

  3. 3.

    Supplementary Information

    This file contains full supplementary table legends for tables 1-9, supplementary results and discussion, full supplementary video legends, and supplementary references.

Zip files

  1. 1.

    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)

  2. 2.

    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.

  3. 3.

    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.

Videos

  1. 1.

    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.

  2. 2.

    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.

  3. 3.

    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.

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DOI

https://doi.org/10.1038/nature26003

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