Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Determining the architectures of macromolecular assemblies

Abstract

To understand the workings of a living cell, we need to know the architectures of its macromolecular assemblies. Here we show how proteomic data can be used to determine such structures. The process involves the collection of sufficient and diverse high-quality data, translation of these data into spatial restraints, and an optimization that uses the restraints to generate an ensemble of structures consistent with the data. Analysis of the ensemble produces a detailed architectural map of the assembly. We developed our approach on a challenging model system, the nuclear pore complex (NPC). The NPC acts as a dynamic barrier, controlling access to and from the nucleus, and in yeast is a 50 MDa assembly of 456 proteins. The resulting structure, presented in an accompanying paper, reveals the configuration of the proteins in the NPC, providing insights into its evolution and architectural principles. The present approach should be applicable to many other macromolecular assemblies.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Determining the architecture of the NPC by integrating spatial restraints from proteomic data.
Figure 2: Structural representation of the NPC.
Figure 3: Protein shape and stoichiometry information.
Figure 4: Localization of proteins by immuno-EM.
Figure 5: Protein interactions of the Nup84 complex.
Figure 6: Protein proximity by affinity purification.
Figure 7: Ambiguity in data interpretation and conditional restraints.
Figure 8: Calculation of the NPC bead structure by satisfaction of spatial restraints.
Figure 9: Bead model, ensemble, localization probability and localization volume.
Figure 10: Ensemble interpretation in terms of protein positions, contacts and configuration.
Figure 11: The structure is increasingly specified by the addition of different types of synergistic experimental information.

Similar content being viewed by others

References

  1. Sali, A., Glaeser, R., Earnest, T. & Baumeister, W. From words to literature in structural proteomics. Nature 422, 216–225 (2003)

    Article  ADS  CAS  PubMed  Google Scholar 

  2. Rout, M. P. et al. The yeast nuclear pore complex: composition, architecture, and transport mechanism. J. Cell Biol. 148, 635–651 (2000)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Macara, I. G. Transport into and out of the nucleus. Microbiol. Mol. Biol. Rev. 65, 570–594 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Weis, K. Nucleocytoplasmic transport: cargo trafficking across the border. Curr. Opin. Cell Biol. 14, 328–335 (2002)

    Article  CAS  PubMed  Google Scholar 

  5. Yang, Q., Rout, M. P. & Akey, C. W. Three-dimensional architecture of the isolated yeast nuclear pore complex: functional and evolutionary implications. Mol. Cell 1, 223–234 (1998)

    Article  CAS  PubMed  Google Scholar 

  6. Beck, M., Lucic, V., Förster, F., Baumeister, E. & Medalia, O. Snapshots of nuclear pore complexes in action captured by cryo-electron tomography. Nature 449, 611–615 (2007)

    Article  ADS  CAS  PubMed  Google Scholar 

  7. Devos, D. et al. Simple fold composition and modular architecture of the nuclear pore complex. Proc. Natl Acad. Sci. USA 103, 2172–2177 (2006)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  8. Havel, T. F. & Wüthrich, K. A distance geometry program for determining the structures of small proteins and other macromolecules from nuclear magnetic resonance measurements of intramolecular 1H–1H proximities in solution. Bull. Math. Biol. 46, 673–698 (1984)

    CAS  MATH  Google Scholar 

  9. Malhotra, A., Tan, R. K. & Harvey, S. C. Prediction of the three-dimensional structure of Escherichia coli 30S ribosomal subunit: a molecular mechanics approach. Proc. Natl Acad. Sci. USA 87, 1950–1954 (1990)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  10. Denning, D. P., Patel, S. S., Uversky, V., Fink, A. L. & Rexach, M. Disorder in the nuclear pore complex: the FG repeat regions of nucleoporins are natively unfolded. Proc. Natl Acad. Sci. USA 100, 2450–2455 (2003)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  11. Lim, R. Y. et al. Flexible phenylalanine-glycine nucleoporins as entropic barriers to nucleocytoplasmic transport. Proc. Natl Acad. Sci. USA 103, 9512–9517 (2006)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  12. Devos, D. et al. Components of coated vesicles and nuclear pore complexes share a common molecular architecture. PLoS Biol. 2, e380 (2004)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Siniossoglou, S. et al. Structure and assembly of the Nup84p complex. J. Cell Biol. 149, 41–54 (2000)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lutzmann, M., Kunze, R., Buerer, A., Aebi, U. & Hurt, E. Modular self-assembly of a Y-shaped multiprotein complex from seven nucleoporins. EMBO J. 21, 387–397 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Strambio-de-Castillia, C., Blobel, G. & Rout, M. P. Isolation and characterization of nuclear envelopes from the Yeast Saccharomyces . J. Cell Biol. 131, 19–31 (1995)

    Article  CAS  PubMed  Google Scholar 

  16. Miller, A. L. et al. Cytoplasmic inositol hexakisphosphate production is sufficient for mediating the Gle1-mRNA export pathway. J. Biol. Chem. 279, 51022–51032 (2004)

    Article  CAS  PubMed  Google Scholar 

  17. Solsbacher, J., Maurer, P., Vogel, F. & Schlenstedt, G. Nup2p, a yeast nucleoporin, functions in bidirectional transport of importin alpha. Mol. Cell. Biol. 20, 8468–8479 (2000)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Marelli, M., Aitchison, J. D. & Wozniak, R. W. Specific binding of the karyopherin Kap121p to a subunit of the nuclear pore complex containing Nup53p, Nup59p, and Nup170p. J. Cell Biol. 143, 1813–1830 (1998)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Archambault, V. et al. Genetic and biochemical evaluation of the importance of Cdc6 in regulating mitotic exit. Mol. Biol. Cell 14, 4592–4604 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Archambault, V. et al. Targeted proteomic study of the cyclin-Cdk module. Mol. Cell 14, 699–711 (2004)

    Article  CAS  PubMed  Google Scholar 

  21. Tackett, A. J. et al. I-DIRT, a general method for distinguishing between specific and nonspecific protein interactions. J. Proteome Res. 4, 1752–1756 (2005)

    Article  CAS  PubMed  Google Scholar 

  22. Cristea, I. M., Williams, R., Chait, B. T. & Rout, M. P. Fluorescent proteins as proteomic probes. Mol. Cell. Proteomics 4, 1933–1941 (2005)

    Article  CAS  PubMed  Google Scholar 

  23. Niepel, M., Strambio-de-Castillia, C., Fasolo, J., Chait, B. T. & Rout, M. P. The nuclear pore complex-associated protein, Mlp2p, binds to the yeast spindle pole body and promotes its efficient assembly. J. Cell Biol. 170, 225–235 (2005)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Cristea, I. M. et al. Tracking and elucidating alphavirus-host protein interactions. J. Biol. Chem. 281, 30269–30278 (2006)

    Article  CAS  PubMed  Google Scholar 

  25. Zhang, W. & Chait, B. T. ProFound: an expert system for protein identification using mass spectrometric peptide mapping information. Anal. Chem. 72, 2482–2489 (2000)

    Article  CAS  PubMed  Google Scholar 

  26. Krutchinsky, A. N., Kalkum, M. & Chait, B. T. Automatic identification of proteins with a MALDI-quadrupole ion trap mass spectrometer. Anal. Chem. 73, 5066–5077 (2001)

    Article  CAS  PubMed  Google Scholar 

  27. Stelter, P. et al. Molecular basis for the functional interaction of dynein light chain with the nuclear-pore complex. Nature Cell Biol. 9, 788–796 (2007)

    Article  CAS  PubMed  Google Scholar 

  28. Murphy, R., Watkins, J. L. & Wente, S. R. GLE2, a Saccharomyces cerevisiae homologue of the Schizosaccharomyces pombe export factor RAE1, is required for nuclear pore complex structure and function. Mol. Biol. Cell 7, 1921–1937 (1996)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Murphy, R. & Wente, S. R. An RNA-export mediator with an essential nuclear export signal. Nature 383, 357–360 (1996)

    Article  ADS  CAS  PubMed  Google Scholar 

  30. Lutzmann, M. et al. Reconstitution of Nup157 and Nup145N into the Nup84 complex. J. Biol. Chem. 280, 18442–18451 (2005)

    Article  CAS  PubMed  Google Scholar 

  31. Bailer, S. M. et al. Nup116p associates with the Nup82p-Nsp1p-Nup159p nucleoporin complex. J. Biol. Chem. 275, 2354–23548 (2000)

    Article  Google Scholar 

  32. Grandi, P., Doye, V. & Hurt, E. C. Purification of NSP1 reveals complex formation with ‘GLFG’ nucleoporins and a novel nuclear pore protein NIC96. EMBO. J. 12, 3061–3071 (1993)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Shen, M. Y. & Sali, A. Statistical potential for assessment and prediction of protein structures. Protein Sci. 15, 2507–2524 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Harding, S. E. Determination of macromolecular homogeneity, shape, and interactions using sedimentation velocity analytical ultracentrifugation. Methods Mol. Biol. 22, 61–73 (1994)

    CAS  PubMed  Google Scholar 

  35. Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E. L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305, 567–580 (2001)

    Article  CAS  PubMed  Google Scholar 

  36. Alber, F., Kim, M. F. & Sali, A. Structural characterization of assemblies from overall shape and subcomplex compositions. Structure 13, 435–445 (2005)

    Article  CAS  PubMed  Google Scholar 

  37. Alber, F. et al. The molecular architecture of the nuclear pore complex. Nature doi: 10.1038/nature06405 (this issue).

  38. Akey, C. W. & Radermacher, M. Architecture of the Xenopus nuclear pore complex revealed by three-dimensional cryo-electron microscopy. J. Cell Biol. 122, 1–19 (1993)

    Article  CAS  PubMed  Google Scholar 

  39. Stoffler, D. et al. Cryo-electron tomography provides novel insights into nuclear pore architecture: implications for nucleocytoplasmic transport. J. Mol. Biol. 328, 119–130 (2003)

    Article  CAS  PubMed  Google Scholar 

  40. Kiseleva, E. et al. Yeast nuclear pore complexes have a cytoplasmic ring and internal filaments. J. Struct. Biol. 145, 272–288 (2004)

    Article  CAS  PubMed  Google Scholar 

  41. Hinshaw, J. E., Carragher, B. O. & Milligan, R. A. Architecture and design of the nuclear pore complex. Cell 69, 1133–1141 (1992)

    Article  CAS  PubMed  Google Scholar 

  42. Beck, M. et al. Nuclear pore complex structure and dynamics revealed by cryoelectron tomography. Science 306, 1387–1390 (2004)

    Article  ADS  CAS  PubMed  Google Scholar 

  43. Pante, N. & Kann, M. Nuclear pore complex is able to transport macromolecules with diameters of about 39 nm. Mol. Biol. Cell 13, 425–434 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  Google Scholar 

  45. Schurmann, G., Haspel, J., Grumet, M. & Erickson, H. P. Cell adhesion molecule L1 in folded (horseshoe) and extended conformations. Mol. Biol. Cell 12, 1765–1773 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank H. Shio for performing the electron microscopic studies; J. Fanghänel, M. Niepel and C. Strambio-de-Castillia for help in developing the affinity purification techniques; M. Magnasco for discussions and advice; A. Kruchinsky for assistance with mass spectrometry; M. Topf, D. Korkin, F. Davis, M.-Y. Shen, F. Foerster, N. Eswar, M. Kim, D. Russel, B. Peterson and B. Webb for many discussions about structure characterization by satisfaction of spatial restraints; C. Johnson, S. G. Parker and C. Silva, T. Ferrin and T. Goddard for preparation of some figures; and S. Pulapura and X. J. Zhou for their help with the design of the conditional diameter restraint. We are grateful to J. Aitchison for discussion and insightful suggestions. We also thank all other members of the Chait, Rout and Sali laboratories for their assistance. We acknowledge support from an Irma T. Hirschl Career Scientist Award (M.P.R.), a Sinsheimer Scholar Award (M.P.R.), a grant from the Rita Allen Foundation (M.P.R.), a grant from the American Cancer Society (M.P.R.), the Sandler Family Supporting Foundation (A.S.), the Human Frontier Science Program (A.S., L.M.V.), NSF (A.S.), and grants from the National Institutes of Health (B.T.C., M.P.R., A.S.), as well as computer hardware gifts from R. Conway, M. Homer, Intel, Hewlett-Packard, IBM and Netapp (A.S.).

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Brian T. Chait, Michael P. Rout or Andrej Sali.

Supplementary information

Supplementary Information

The file contains Supplementary Methods, Supplementary Figures 1-27, Supplementary Tables 1-10 and additional references. (PDF 8230 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Alber, F., Dokudovskaya, S., Veenhoff, L. et al. Determining the architectures of macromolecular assemblies. Nature 450, 683–694 (2007). https://doi.org/10.1038/nature06404

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature06404

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing