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Structural Analysis of Multiprotein Complexes by Cross-linking, Mass Spectrometry, and Database Searching*

https://doi.org/10.1074/mcp.M700274-MCP200Get rights and content
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Most protein complexes are inaccessible to high resolution structural analysis. We report the results of a combined approach of cross-linking, mass spectrometry, and bioinformatics to two human complexes containing large coiled-coil segments, the NDEL1 homodimer and the NDC80 heterotetramer. An important limitation of the cross-linking approach, so far, was the identification of cross-linked peptides from fragmentation spectra. Our novel approach overcomes the data analysis bottleneck of cross-linking and mass spectrometry. We constructed a purpose-built database to match spectra with cross-linked peptides, define a score that expresses the quality of our identification, and estimate false positive rates. We show that our analysis sheds light on critical structural parameters such as the directionality of the homodimeric coiled coil of NDEL1, the register of the heterodimeric coiled coils of the NDC80 complex, and the organization of a tetramerization region in the NDC80 complex. Our approach is especially useful to address complexes that are difficult in addressing by standard structural methods.

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Published, MCP Papers in Press, October 5, 2007, DOI 10.1074/mcp.M700274-MCP200

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This work was supported by the European Community through a Marie Curie Excellence Grant and the Italian Association for Cancer Research (AIRC). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.

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These authors contributed equally to this work.

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Present address: Howard Hughes Medical Inst./University of California, 742 Stanley Hall, MS 3220, Berkeley, CA 94720-3220.