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Bacterial Protein Secretion Systems pp 377–413Cite as

Structural Analysis of Protein Complexes by Cryo Electron Microscopy

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1615))

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.

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Acknowledgments

The authors thank Dr. H. White for reading the manuscript and useful discussions that led to improvements in the manuscript. This work was funded by MRC Grant MR/K012401/1 to E.V.O. The authors apologise for not covering all methods fully owing to space constraints.

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  1. Institute for Structural and Molecular Biology, School of Biological Sciences, Birkbeck College, London, WC1E 7HX, UK

    Tiago R. D. Costa, Athanasios Ignatiou & Elena V. Orlova

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  1. Tiago R. D. Costa
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  2. Athanasios Ignatiou
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  3. Elena V. Orlova
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Correspondence to Elena V. Orlova .

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  1. Institut de Microbiologie de la Méditerranée, Aix-Marseille Univ - CNRS, Marseille, France

    Laure Journet

  2. Institut de Microbiologie de la Méditerranée, Aix-Marseille Univ - CNRS, Marseille, France

    Eric Cascales

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Costa, T.R.D., Ignatiou, A., Orlova, E.V. (2017). Structural Analysis of Protein Complexes by Cryo Electron Microscopy. In: Journet, L., Cascales, E. (eds) Bacterial Protein Secretion Systems. Methods in Molecular Biology, vol 1615. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7033-9_28

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