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Technology Topics Hybrid/Integrative Methods

Nuclear Pore Complex: Higher Resolution of Macromolecules

SBKB [doi:10.1038/sbkb.2014.244]
Technical Highlight - February 2015
Short description: Integrating mass spectrometry data for crosslinked oligomers reveals details of the yeast nuclear pore complex.

DSS (green) and EDC (red) crosslinks provided complementary spatial information in the analysis of the Nup84 complex. Figure from ref. 1 , © 2014 The American Society for Biochemistry and Molecular Biology.

When classical structural determination methods fail to provide high-resolution information for large complexes that are often dynamic and flexible, integrative approaches can reveal more details. Available data of various resolutions, obtained using a range of methods, are used to generate spatial restraints, and structures that satisfy these restraints are calculated and analyzed. Chemical crosslinking followed by mass spectrometry (CX-MS) facilitates determination of spatial restraints, and researchers have now improved on the approach by using two complementary crosslinkers.

The Chait, Rout and Sali groups optimized the structural characterization pipeline. Using established methods, they affinity purified the endogenously tagged ∼600 kDa Nup84 complex in a proof-of-principle study. Comprised of seven nucleoporins, the Nup84 complex forms the outer rings of the nuclear pore complex. The authors crosslinked natively purified complexes with disuccinimidyl suberate (DSS), which targets primary amines, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), which targets more abundant carboxylic groups. After digests, they separated the resulting peptides by size-exclusion chromatography and analyzed them by liquid chromatography-MS; a pLink search identified crosslinks. DSS and EDC crosslinks provided complementary restraints, and the data were mutually confirmatory. The authors suggest that some of the differences could reflect heterogeneity and/or conformational flexibility in the complexes, and disorder previously observed by electron microscopy (EM).

In addition to these experimental data, in the modeling, the researchers integrated restraints based on EM analyses of Nup84, X-ray crystallography data for domains of the complex as well as comparative modeling data. Of the possible configurations, 6,520 models satisfied the restraints, and the researchers clustered and analyzed these solutions to come up with final models. The highest resolution achieved for one region of the complex was 12.7 Å. The reported structure revealed greater detail, particularly in the hub region of the complex, which showed how constituent proteins interact.

Irene Kaganman

References

  1. Y. Shi et al. Structural characterization by crosslinking reveals the detailed architecture of a coatomer-related heptameric module from the nuclear pore complex.
    Mol Cell Proteomics 13, 2927-2943 (2014). doi:10.1074/mcp.M114.041673

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