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    Structural analysis of protein complexes by cryo electron microscopy

    Costa, Tiago and Ignatiou, Athenasios and Orlova, Elena (2017) Structural analysis of protein complexes by cryo electron microscopy. Methods in Molecular Biology 1615 , pp. 377-413. ISSN 1064-3745.

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    Abstract

    Structural studies of biocomplexes using single-particle cryo-electron microscopy (cryo-EM) is now a well-established technique in structural biology and has become competitive with X-ray crystallography. The latest advances in EM enable us to determine structures of protein complexes at 3–5 Å resolution for an extremely broad range of sizes from ~200 kDa up to hundreds of megadaltons (Bartesaghi et al., Science 348(6239):1147–1151, 2051; Bai et al., Nature 525(7568):212–217, 2015; Vinothkumar et al., Nature 515(7525):80–84, 2014; Grigorieff and Harrison, Curr Opin Struct Biol 21(2):265–273, 2011). The majority of biocomplexes comprise a number of different components and are not amenable to crystallisation. Secretion systems are typical examples of such multi-protein complexes, and structural studies of them are extremely challenging. The only feasible approach to revealing their spatial organisation and functional modification is cryo-EM. The development of systems for digital registration of images and algorithms for the fast and efficient processing of recorded images and subsequent analysis facilitated the determination of structures at near-atomic resolution. In this review we will describe sample preparation for cryo-EM, how data are collected by new detectors, and the logistics of image analysis through the basic steps required for reconstructions of both small and large biological complexes and their refinement to nearly atomic resolution. The processing workflow is illustrated using examples of EM analysis of a Type IV Secretion System.

    Metadata

    Item Type: Article
    Keyword(s) / Subject(s): Cryo-electron microscopy, Sample preparation, Single particle analysis, Image processing, Type IV secretion system
    School: Birkbeck Schools and Departments > School of Science > Biological Sciences
    Depositing User: Administrator
    Date Deposited: 18 Aug 2017 09:17
    Last Modified: 18 Aug 2017 09:17
    URI: http://eprints.bbk.ac.uk/id/eprint/19424

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