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Determining the architectures of macromolecular assemblies

Nature volume 450, pages 683–694 (2007)Cite this article

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.

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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.

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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.).

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Author notes

  1. Svetlana Dokudovskaya, Liesbeth M. Veenhoff, Julia Kipper, Damien Devos, Adisetyantari Suprapto & Orit Karni-Schmidt

    Present address: Present addresses: Laboratory of Nucleocytoplasmic Transport, Institut Jacques Monod, 2 place Jussieu, Tour 43, Paris 75251, France (S.D.); Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands (L.M.V.); German Aerospace Center (PT-DLR), Heinrich-Konen-Strasse 1, D-53227 Bonn, Germany (J.K.); Structural Bioinformatics, EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany (D.D.); Office of Technology Transfer, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA (A.S.); Herbert Irving Comprehensive Cancer Center, Columbia University, 1130 St Nicholas Avenue, New York, New York 10032, USA (O.K.-S.)., New York

  2. Frank Alber, Svetlana Dokudovskaya and Liesbeth M. Veenhoff: 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, Byers Hall, Suite 503B, 1700 4th Street, University of California at San Francisco, San Francisco, California 94158-2330, USA, California

    Frank Alber, Damien Devos & Andrej Sali

  2. Laboratory of Cellular and Structural Biology, and,, California

    Svetlana Dokudovskaya, Liesbeth M. Veenhoff, Julia Kipper, Adisetyantari Suprapto, Orit Karni-Schmidt, Rosemary Williams & Michael P. Rout

  3. Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA , New York

    Wenzhu Zhang & Brian T. Chait

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

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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

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