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In situ structural analysis of the human nuclear pore complex

Abstract

Nuclear pore complexes are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Determining their 110-megadalton structure imposes a formidable challenge and requires in situ structural biology approaches. Of approximately 30 nucleoporins (Nups), 15 are structured and form the Y and inner-ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ~60 nm in diameter1. The scaffold is decorated with transport-channel Nups that often contain phenylalanine-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here we combine cryo-electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modelling to generate, to our knowledge, the most comprehensive architectural model of the human nuclear pore complex to date. Our data suggest previously unknown protein interfaces across Y complexes and to inner-ring complex members. We show that the transport-channel Nup358 (also known as Ranbp2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport-channel Nups. We conclude that, similar to coated vesicles, several copies of the same structural building block—although compositionally identical—engage in different local sets of interactions and conformations.

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Figure 1: Tomographic map of the human NPC.
Figure 2: Nup358 complex stabilizes the CR.
Figure 3: Scaffold architecture of the human NPC.

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References

  1. 1

    Hoelz, A., Debler, E. W. & Blobel, G. The structure of the nuclear pore complex. Annu. Rev. Biochem. 80, 613–643 (2011)

    CAS  Article  Google Scholar 

  2. 2

    Vollmer, B. & Antonin, W. The diverse roles of the Nup93/Nic96 complex proteins – structural scaffolds of the nuclear pore complex with additional cellular functions. Biol. Chem. 395, 515–528 (2014)

    CAS  Article  Google Scholar 

  3. 3

    Hurt, E. & Beck, M. Towards understanding nuclear pore complex architecture and dynamics in the age of integrative structural analysis. Curr. Opin. Cell Biol. 34, 31–38 (2015)

    CAS  Article  Google Scholar 

  4. 4

    Bui, K. H. et al. Integrated structural analysis of the human nuclear pore complex scaffold. Cell 155, 1233–1243 (2013)

    CAS  Article  Google Scholar 

  5. 5

    Kampmann, M. & Blobel, G. Three-dimensional structure and flexibility of a membrane-coating module of the nuclear pore complex. Nature Struct. Mol. Biol. 16, 782–788 (2009)

    CAS  Article  Google Scholar 

  6. 6

    Thierbach, K. et al. Protein interfaces of the conserved Nup84 complex from Chaetomium thermophilum shown by crosslinking mass spectrometry and electron microscopy. Structure 21, 1672–1682 (2013)

    CAS  Article  Google Scholar 

  7. 7

    Fornerod, M., van Baal, S., Valentine, V., Shapiro, D. N. & Grosveld, G. Chromosomal localization of genes encoding CAN/Nup214-interacting proteins–human CRM1 localizes to 2p16, whereas Nup88 localizes to 17p13 and is physically linked to SF2p32. Genomics 42, 538–540 (1997)

    CAS  Article  Google Scholar 

  8. 8

    Werner, A., Flotho, A. & Melchior, F. The RanBP2/RanGAP1*SUMO1/Ubc9 complex is a multisubunit SUMO E3 ligase. Mol. Cell 46, 287–298 (2012)

    CAS  Article  Google Scholar 

  9. 9

    Ori, A. et al. Cell type-specific nuclear pores: a case in point for context-dependent stoichiometry of molecular machines. Mol. Syst. Biol. 9, 648 (2013)

    CAS  Article  Google Scholar 

  10. 10

    Stuwe, T. et al. Nuclear pores. Architecture of the nuclear pore complex coat. Science 347, 1148–1152 (2015)

    ADS  CAS  Article  Google Scholar 

  11. 11

    Bilokapic, S. & Schwartz, T. U. Molecular basis for Nup37 and ELY5/ELYS recruitment to the nuclear pore complex. Proc. Natl Acad. Sci. USA 109, 15241–15246 (2012)

    ADS  CAS  Article  Google Scholar 

  12. 12

    Seo, H. S. et al. Structural and functional analysis of Nup120 suggests ring formation of the Nup84 complex. Proc. Natl Acad. Sci. USA 106, 14281–14286 (2009)

    ADS  CAS  Article  Google Scholar 

  13. 13

    Nagy, V. et al. Structure of a trimeric nucleoporin complex reveals alternate oligomerization states. Proc. Natl Acad. Sci. USA 106, 17693–17698 (2009)

    ADS  CAS  Article  Google Scholar 

  14. 14

    Alber, F. et al. The molecular architecture of the nuclear pore complex. Nature 450, 695–701 (2007)

    ADS  CAS  Article  Google Scholar 

  15. 15

    Boehmer, T., Jeudy, S., Berke, I. C. & Schwartz, T. U. Structural and functional studies of Nup107/Nup133 interaction and its implications for the architecture of the nuclear pore complex. Mol. Cell 30, 721–731 (2008)

    CAS  Article  Google Scholar 

  16. 16

    Glavy, J. S. et al. Cell-cycle-dependent phosphorylation of the nuclear pore Nup107–160 subcomplex. Proc. Natl Acad. Sci. USA 104, 3811–3816 (2007)

    ADS  CAS  Article  Google Scholar 

  17. 17

    Laurell, E. et al. Phosphorylation of Nup98 by multiple kinases is crucial for NPC disassembly during mitotic entry. Cell 144, 539–550 (2011)

    CAS  Article  Google Scholar 

  18. 18

    Shi, Y. et al. Structural characterization by cross-linking reveals the detailed architecture of a coatomer-related heptameric module from the nuclear pore complex. Mol. Cell. Proteomics 13, 2927–2943 (2014)

    CAS  Article  Google Scholar 

  19. 19

    Culjkovic-Kraljacic, B., Baguet, A., Volpon, L., Amri, A. & Borden, K. L. The oncogene eIF4E reprograms the nuclear pore complex to promote mRNA export and oncogenic transformation. Cell Rep. 2, 207–215 (2012)

    CAS  Article  Google Scholar 

  20. 20

    Kim, D. I. et al. Probing nuclear pore complex architecture with proximity-dependent biotinylation. Proc. Natl Acad. Sci. USA 111, E2453–E2461 (2014)

    CAS  Article  Google Scholar 

  21. 21

    Flemming, D. et al. Analysis of the yeast nucleoporin Nup188 reveals a conserved S-like structure with similarity to karyopherins. J. Struct. Biol. 177, 99–105 (2012)

    CAS  Article  Google Scholar 

  22. 22

    Theerthagiri, G., Eisenhardt, N., Schwarz, H. & Antonin, W. The nucleoporin Nup188 controls passage of membrane proteins across the nuclear pore complex. J. Cell Biol. 189, 1129–1142 (2010)

    CAS  Article  Google Scholar 

  23. 23

    Damelin, M. & Silver, P. A. In situ analysis of spatial relationships between proteins of the nuclear pore complex. Biophys. J. 83, 3626–3636 (2002)

    ADS  CAS  Article  Google Scholar 

  24. 24

    Gaik, M. et al. Structural basis for assembly and function of the Nup82 complex in the nuclear pore scaffold. J. Cell Biol. 208, 283–297 (2015)

    Article  Google Scholar 

  25. 25

    Zemp, I. et al. Distinct cytoplasmic maturation steps of 40S ribosomal subunit precursors require hRio2. J. Cell Biol. 185, 1167–1180 (2009)

    CAS  Article  Google Scholar 

  26. 26

    Ori, A., Andres-Pons, A. & Beck, M. The use of targeted proteomics to determine the stoichiometry of large macromolecular assemblies. Methods Cell Biol. 122, 117–146 (2014)

    CAS  Article  Google Scholar 

  27. 27

    Wessel, D. & Flugge, U. I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138, 141–143 (1984)

    CAS  Article  Google Scholar 

  28. 28

    Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005)

    Article  Google Scholar 

  29. 29

    Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nature Methods 10, 584–590 (2013)

    CAS  Article  Google Scholar 

  30. 30

    Schur, F. K., Hagen, W. J., de Marco, A. & Briggs, J. A. Determination of protein structure at 8.5 Å resolution using cryo-electron tomography and sub-tomogram averaging. J. Struct. Biol. 184, 394–400 (2013)

    CAS  Article  Google Scholar 

  31. 31

    Movassagh, T., Bui, K. H., Sakakibara, H., Oiwa, K. & Ishikawa, T. Nucleotide-induced global conformational changes of flagellar dynein arms revealed by in situ analysis. Nature Struct. Mol. Biol. 17, 761–767 (2010)

    CAS  Article  Google Scholar 

  32. 32

    Kosinski, J., Barbato, A. & Tramontano, A. MODexplorer: an integrated tool for exploring protein sequence, structure and function relationships. Bioinformatics 29, 953–954 (2013)

    CAS  Article  Google Scholar 

  33. 33

    Guex, N. & Peitsch, M. C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723 (1997)

    CAS  Article  Google Scholar 

  34. 34

    Kurowski, M. A. & Bujnicki, J. M. GeneSilico protein structure prediction meta-server. Nucleic Acids Res. 31, 3305–3307 (2003)

    CAS  Article  Google Scholar 

  35. 35

    Eswar, N. et al. Comparative protein structure modeling using MODELLER. Current Proc. Protein Sci. Ch. 2 (2007)

  36. 36

    Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

    CAS  Article  Google Scholar 

  37. 37

    Whittle, J. R. & Schwartz, T. U. Architectural nucleoporins Nup157/170 and Nup133 are structurally related and descend from a second ancestral element. J. Biol. Chem. 284, 28442–28452 (2009)

    CAS  Article  Google Scholar 

  38. 38

    Suhre, K. & Sanejouand, Y. H. ElNemo: a normal mode web server for protein movement analysis and the generation of templates for molecular replacement. Nucleic Acids Res. 32, W610–W614 (2004)

    CAS  Article  Google Scholar 

  39. 39

    de Vries, S. J. et al. HADDOCK versus HADDOCK: new features and performance of HADDOCK2.0 on the CAPRI targets. Proteins 69, 726–733 (2007)

    CAS  Article  Google Scholar 

  40. 40

    Minguez, P. et al. PTMcode v2: a resource for functional associations of post-translational modifications within and between proteins. Nucleic Acids Res. 43, D494–D502 (2015)

    CAS  Article  Google Scholar 

  41. 41

    Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nature Methods 11, 63–65 (2014)

    CAS  Article  Google Scholar 

  42. 42

    Walther, T. C. et al. The cytoplasmic filaments of the nuclear pore complex are dispensable for selective nuclear protein import. J. Cell Biol. 158, 63–77 (2002)

    CAS  Article  Google Scholar 

  43. 43

    Drin, G. et al. A general amphipathic α-helical motif for sensing membrane curvature. Nature Struct. Mol. Biol. 14, 138–146 (2007)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We are grateful to W. Baumeister and J. Plitzko for access to the electron microscopy facility of the Max Planck Institute of Biochemistry. We thank F. Schur, K. Beck, J. Briggs, C. Sachse, E. Hurt and F. Melchior for their critical advice and A. Neal for critical reading of the manuscript. We gratefully acknowledge support from EMBL’s mechanical workshop, the Electron Microscopy and Proteomics Core Facilities, the Centre for Statistical Data Analysis and thank J. Krijgsveld, J. Kirkpatrick and B. Klaus. K.H.B. was supported by postdoctoral fellowships from the Swiss National Science Foundation, the European Molecular Biology Organization and Marie Curie Actions. A.O. was supported by postdoctoral fellowships from the Alexander von Humboldt Foundation and Marie Curie Actions. A.L.D. was supported by the Robert Crooks Stanley Fellowship at the Stevens Institute of Technology and National Institute on Aging (NIA) grant 1R21AG047433-01. J.K. was supported by the EMBL Interdisciplinary Postdoc Programme under Marie Curie COFUND Actions. J.S.G. was supported by an Ignition Grant Initiative from Stevens Institute of Technology and NIA grant 1R21AG047433-01. M.B. acknowledges funding by EMBL and the European Research Council (309271-NPCAtlas).

Author information

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Authors

Contributions

A.v.A., J.K. and W.A. designed and performed experiments, analysed data and wrote the manuscript. A.v.A., J.K. and P.K. performed modelling. B.V., L.S., A.O., A.L.D., M.-T.M., K.B., W.H., A.A.-P., N.B. and S.M. designed and performed experiments, and analysed data. L.P. analysed data and performed modelling. J.S.G., E.A.L. and P.B. designed experiments and oversaw the project. K.H.B. designed and performed experiments, analysed data, oversaw the project and wrote the manuscript. M.B. designed experiments, analysed data, oversaw the project and wrote the manuscript.

Corresponding authors

Correspondence to Khanh Huy Bui or Martin Beck.

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The authors declare no competing financial interests.

Additional information

Associated with this manuscript are Electron Microscopy Data Bank entries EMD-3103, EMD-3104, EMD-3105, EMD-3106 and EMD-3107 and Protein Data Bank entry 5A9Q.

Extended data figures and tables

Extended Data Figure 1 Tomographic map of the human NPC.

a, Orthoslices through the nucleocytoplasmic axis, CR, IR and NR of a tomographic structure of the human NPC obtained using a direct electron detector (this study) compared to a structure obtained with a conventional detector using a similar experimental workflow (Electron Microscopy Database accession number EMD-2444). In both cases, the CR, IR and NR were aligned independently. The arrowhead indicates a transmembrane domain that is resolved in the IR. b, Fourier shell correlation curves of the CR, IR and NR regions. c, Histogram corresponding to the colour-coded local resolution map shown in d that was calculated using ResMap41. d, A single segment of the NR ring is shown (in all other figures the segments are shown jointly with their anterior and/or posterior asymmetric unit) at two different isosurface thresholds. The redundant density of the outer Nup43 β-propeller (horizontal arrowhead) and the Nup133 middle domain (vertical arrowhead) of two neighbouring asymmetric units are indicated for orientation. The reduced resolution at the edges is due to the border of the mask used during alignment and averaging that covered about 1.5 times the asymmetric unit.

Extended Data Figure 2 B-factor correction.

X-ray structures filtered to the overall resolution of the tomographic map were compared to tomographic maps corrected with different B-factors to choose an appropriate B-factor. a, X-ray structure of Nup107–Nup96–Sec13 (top), filtered to 23 Å (below) as compared to respective regions of the outer vertex corrected with B-factors ranging from 2,000–9,000 Å2. b, B-factors of 6,000–8,000 Å2 most realistically resemble features of the X-ray structures. Three regions of the tomographic map are superimposed with the respective X-ray structures at B-factors of 6,000 Å2 in comparison to 8,000 Å2. In these well-resolved regions, additional features such as more detailed shapes of β-propellers or the Nup107 finger domain (see also Fig. 2a, b and Extended Data Fig. 4e) are apparent at 8,000 Å2. Owing to local deviations in resolution (Extended Data Fig. 1c, d) a more conservative B-factor of 6,000 Å2 was chosen to correct the averages. Asterisks indicate structures that can be unambiguously positioned but have some uncertainty in their orientation, that is the Nup85 carboxy-terminal domain (CTD). c, Systematic fitting of the X-ray structure of Nup107–Nup96–Sec13 into the tomographic map as shown in Extended Data Fig. 7a but at different B-factors. Adjusted P values are shown ranked; the four true positive hits are shown in red in the inset. The latter are consistently identified as top hits, except when a B-factor of 9,000 Å2 is used.

Extended Data Figure 3 Comparison of hybrid model of the Y complex to the X-ray structure of the vertex region10.

The hybrid model of the Y complex (NR) shown on the left side was generated independently from the coordinates X-ray structure of the vertex shown on the right side. The structures of Y complex members were fitted into the tomographic map based on spatial restraints and complementary information (see Supplementary Table 1 for details). The outer and inner Y complexes/vertices are shown separately on the top and the bottom. The molecular weight of one human Y complex is approximately 1 MDa, the majority of which is structured. Asterisks indicate structures that can be unambiguously positioned but have some uncertainty in their orientation. Those are the β-propellers of Elys and Nup133 as well as Nup85-CTD.

Extended Data Figure 4 The inner and outer Y complexes have distinct conformations and engage in locally specific sets of interactions.

a, Arrangement of the inner and outer Y complex as seen from above. X-ray structures were filtered to 2.3 nm resolution and coloured by protein. Positions of the five interfaces between Y complexes are indicated. b, The inner and outer copies of Nup160 assume the same normal vector with respect to the membrane and are slightly tilted to each other because of the different diameters from the central axis. c, The hinges between the Nup107 C- and N-terminal domains as well as within the Nup133 C-terminal domain have a different conformation in the inner and outer Y complex. In the case of the inner stem, the middle domain of Nup133 appears slightly bent inwards compared to the conformation revealed in the X-ray structure, which can be accounted for by introducing a hinge, as previously predicted based on the Nup133 structure37. d, Magnified views showing details of five interfaces (I–V). The panels on the right indicate the viewing point. (I) Sec13 of the outer vertex interfaces with the Nup107 N-terminal domain of the inner vertex4,10; (II) the N-terminal β-propeller of Nup133 interfaces with Nup160 of the posterior asymmetric unit in the case of both Y complexes, forming the head-to-tail contact that facilitates ring formation12,14; (III) therefore, only the β-propeller of the inner copy of Nup133 is sandwiched between both Nup160 proteins. In this case, the N-terminal α-helical domain extends into a larger interface with the outer Nup160 of the posterior asymmetric unit; (IV) the C terminus of the inner Nup133 branches out of the inner stem to form a contact with its counterpart on the outer stem. This relatively small interface is reminiscent of a crystal contact observed in the Nup133–Nup107 structure (Protein Data Bank accession number 3I4R)37; and (V) Nups 85 and 43 of the outer vertex form an interface with the C-terminal domain of Nup107 of the inner stem. e, The Nup107 finger domain (red) is exclusive to higher eukaryotes. While its inner copy (shown) interfaces with Nup43 and Nup85 of the outer vertex, its outer copy interfaces with the density connecting both Y complexes (Fig. 2a, b). A phosphorylation site in the finger domain is depicted as dark-red spheres. Asterisks indicate structures that can be unambiguously positioned but have some uncertainty in their orientation, that is Nup85-CTD. f, Vertex region of the CR as seen from the central channel. Phosphorylation sites are represented as in e. The density connecting both Y complexes is segmented as in Fig. 2b and is in close contact with several phosphorylation sites in Sec13, Nup96 and Nup107. Phosphorylation sites in Seh1 and Nup85 are in direct proximity to the Nup214 complex region.

Extended Data Figure 5 Connection of the inner and outer Y complexes.

a, Four clamp-shaped densities (segmented red) emanate from the Nup96/107 region of both Y complexes in the CR. Only the inner clamps connect both Y complexes, whereas the outer ones protrude into a more complex substructure at the outer periphery of the CR (Fig. 2b). b, Same as in Fig. 2c, d but for the NR. c, Volcano plot visualizing shotgun proteomics data obtained of HeLa cells in the Nup358 knockdown (treatment) as compared to the control condition. d, Nuclear transport assays of NLS–MBP–GFP (that is, a nuclear localization sequence bound to a GFP-tagged maltose-binding protein) in non-treated cells and the Nup358 knockdown condition. Cargos with a classical nuclear localization signal are imported in the absence of Nup358 as previously observed42, although a lower efficiency cannot be excluded. e, Classification of sub-tomograms of the knockdown condition reveals that approximately 5% of all asymmetric units contain an outer Y complex in the CR, which is in excellent agreement with the knockdown efficiency of 95%. Classification was done on the level of asymmetric units. Transferred to the level of NPCs, it suggests that out of 920 NPCs observed under gene silencing conditions, 663 had no outer Y complex in the CR, 183 had one, 49 had two, 14 had three, 4 had four, 6 had five, 0 had six, 1 had seven and 0 had eight. f, The observed adjacency of outer Y complexes in the CR under knockdown conditions was much higher than expected. The 5% of asymmetric units that contained outer Y complexes in the CR were analysed on the NPC level to determine whether their neighbouring asymmetric units also contained outer Y complexes in the CR. The observed frequency of adjacency is shown in dark blue. The respective total number of observed outer Y complexes in the CR and the number of the ones that had adjacent partners is indicated as (n/m). The observed frequency is considerably elevated over the theoretical frequency (shown in bright blue) that would be expected if Y complexes would bind to random subunits. This observation implies cooperativity for Y complex assembly/maintenance within the CR that might arise through the head-to-tail contact of adjacent Y complexes. NPCs with zero, one and eight outer Y complexes per CR are not shown because they cannot contain any adjacent Y complexes or were not observed.

Extended Data Figure 6 Structural signatures of inner-ring scaffold Nups and membrane-binding motifs of Nup160 and Nup133.

a, Same as Fig. 3a but for the CR. b, Fits of Nup160 (left) of the outer (orange) and inner (grey) Y complexes into the NR are shown. Additional density accounting for the C-terminal domain of Elys is indicated. Fits of Nup133 are shown at normal (centre) and high (right) isosurface thresholds. At the higher isosurface threshold, density linking both Nup133 domains is also apparent in the outer stem. The membrane-binding motifs43 are coloured red. Asterisks indicate structures that can be unambiguously positioned but have some uncertainty in their orientation. Those are the β-propellers of Elys and Nup133 as well as Nup85-CTD.

Extended Data Figure 7 Systematic fitting of selected NPC components to the electron microscopy map.

Each panel shows the 20 best-scoring fits (left), a plot of P values for all the solutions (right) and the top solutions in the inset. The models used for fitting are shown as ribbon representation. The fits and data points are coloured according to the groups with similar P value ranges. The group of fits with the best P values is coloured red (high-confidence fits), the second-best group (medium-confidence fits) is coloured blue, and all remaining are coloured cyan. The membrane density has been removed for clarity. a, The Sec13–Nup96–Nup107 subcomplex, for which the ground-truth is known because it is part of the vertex. bd, same as a but for the N-terminal domains of Nup155, the open conformation of Nup205/188-NTD (template Nup205) and the closed conformation of Nup205/188-NTD (template Nup188), respectively. Solution id, solution identity.

Extended Data Figure 8 Co-elution analysis to detect weak nucleoporin interactions.

To detect weak interactions of scaffold nucleoporins we combined rapid affinity isolation with gel filtration and quantitative targeted proteomics to measure absolute protein abundances. HEK293 cells expressing various affinity-tagged Nups (in contrast to Fig. 3c without nocodazole arrest) were lysed using mild conditions and sonication for protein solubilization. Affinity isolates were subjected to gel-filtration and all fractions were analysed using targeted mass spectrometry, as previously described24,26, to measure protein abundances in the high- and low-molecular-weight fractions. The high-molecular-weight fractions are indicative of potential outgoing interactions of large molecular species. The low-molecular-weight fraction will highlight smaller fragments that occur after sonication. a, In the case of affinity-tagged Nup85 the top panel shows the 215 nm absorption curve of the gel-filtration experiment. The middle panel shows the arbitrary protein abundance units of Y-complex members in all fractions (red for Nup85, black for all other Y-complex members). Protein abundances (normalized to the affinity-tagged protein) in the high-molecular-weight fractions (blue bar) and low-molecular-weight fraction (green bar) are shown as bar charts in the bottom panel. The low-molecular-weight peak corresponds to the small arm proteins (Nup85, Seh1 and Nup43), the high-molecular-weight peak to the intact Y complex. b, The same approach was applied to Nup205, Nup93 and Nup155. The seven most abundant co-eluting Nups are shown for the high-molecular-weight fractions (blue bar plots) and low-molecular-weight fractions (green bar plots). In case of Nup85 (top) the seven most abundant proteins apart from the Y-complex members are shown. Weak interactions of Y-complex members with Nup205 and Nup93 are apparent. In the case of Nup155, weak interactions are detected with CR and NR members, as well as Sec13 that localizes to the proximity of the C-terminal domain of Nup155 when fitted into the density connecting the IR with CR/NR. Tpr was excluded from this analysis since it was present in all fractions. Protein abundances based on single reference peptides are marked with an asterisk. c, Same as a (top panel) but for Nup188 affinity purified from nocodazole-arrested cells. d, Same as Fig. 3c but for Nup188 affinity purified from nocodazole-arrested cells. Co-eluting species in the high-molecular-weight fraction are similar to the ones observed for affinity-purified Nup62 (Fig. 3c). Isostoichiometric amounts of Nup188, Nup98, Nup93, Nup62 and an enrichment of Nup214, Nup88 and Rae1 were detected. The Nup188–Nup93 heterodimer thus binds to Nups that are well-established components of the CR, which is consistent with the systematic fitting approach. e, Same as c but corresponding to Fig. 3c. f, Same as b (second panel for Nup205) but for nocodazole-arrested cells. In the case of Nup205, the co-purifying species are similar in nocodazole-arrested as compared to untreated cells.

Extended Data Table 1 Template structures used for homology modelling or selected human crystal structures

Supplementary information

Supplementary information

This file contains Supplementary Table 1 and additional references. (PDF 1548 kb)

Structural analysis of the Y-complex in situ

A segment of the NR is shown isosurface rendered. High-resolution structures are shown superimposed; color code as in Fig. 1 and 3e. (MP4 29488 kb)

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von Appen, A., Kosinski, J., Sparks, L. et al. In situ structural analysis of the human nuclear pore complex. Nature 526, 140–143 (2015). https://doi.org/10.1038/nature15381

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