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Protein Complex Assembly pp 29–44Cite as

High-Throughput Electron Cryo-tomography of Protein Complexes and Their Assembly

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1764))

Abstract

Electron cryo-tomography and subtomogram averaging enable visualization of protein complexes in situ, in three dimensions, in a near-native frozen-hydrated state to nanometer resolutions. To achieve this, intact cells are vitrified and imaged over a range of tilts within an electron microscope. These images can subsequently be reconstructed into a three-dimensional volume representation of the sample cell. Because complexes are visualized in situ, crucial insights into their mechanism, assembly process, and dynamic interactions with other proteins become possible. To illustrate the electron cryo-tomography workflow for visualizing protein complexes in situ, we describe our workflow of preparing samples, imaging, and image processing using Leginon for data collection, IMOD for image reconstruction, and PEET for subtomogram averaging.

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Acknowledgment

LH was supported by a Biotechnology and Biological Sciences Research Council postgraduate training award and Biotechnology and Biological Sciences Research Council Grant BB/L023091/1 to MB.

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Authors and Affiliations

  1. Department of Life Sciences, Imperial College of London, London, UK

    Louie D. Henderson & Morgan Beeby

Authors
  1. Louie D. Henderson
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  2. Morgan Beeby
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Corresponding author

Correspondence to Morgan Beeby .

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Editors and Affiliations

  1. MRC Human Genetics Unit, Institute of Genetics & Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom

    Joseph A. Marsh

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Henderson, L.D., Beeby, M. (2018). High-Throughput Electron Cryo-tomography of Protein Complexes and Their Assembly. In: Marsh, J. (eds) Protein Complex Assembly. Methods in Molecular Biology, vol 1764. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7759-8_2

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  • DOI: https://doi.org/10.1007/978-1-4939-7759-8_2

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7758-1

  • Online ISBN: 978-1-4939-7759-8

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