Determining the architectures of macromolecular assemblies.

Department of Bioengineering and Therapeutic Sciences, 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.
Nature (Impact Factor: 42.35). 12/2007; 450(7170):683-94. DOI: 10.1038/nature06404
Source: PubMed

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.

  • Biophysical Reviews 01/2015; DOI:10.1007/s12551-014-0155-1
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    Advances in Protein Chemistry and Structural Biology 01/2014; 96:77-111. DOI:10.1016/bs.apcsb.2014.06.008 · 3.74 Impact Factor
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    ABSTRACT: The nuclear pore complex (NPC) is one of the largest supramolecular structures in eukaryotic cells. Its octagonal ring-scaffold perforates the nuclear envelope and features a unique molecular machinery that regulates nucleocytoplasmic transport. NPCs are composed of ~30 different nucleoporins (Nups), averaged at 8, 16 or 32 copies per NPC. This estimate has not been confirmed for individual NPCs in living cells due to the inherent difficulty of counting proteins inside single supramolecular complexes. Here we used single-molecule SPEED microscopy to directly count the copy-number of twenty-four different Nups within individual NPCs of live yeast, and found agreement as well as significant deviation from previous estimates. As expected, we counted 8 copies of four peripheral Nups and 16 copies of fourteen scaffold Nups. Unexpectedly, we counted a maximum of 16 copies of Nsp1 and Nic96, rather than 32 as previously estimated; and found only 10–15 copies of six other Nups, rather than 8 or 16 copies as expected. This in situ molecular-counting technology can test structure-function models of NPCs and other supramolecular structures in cells.
    Scientific Reports 03/2015; 5. DOI:10.1038/srep09372 · 5.08 Impact Factor

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