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Protein Nanotechnology pp 221–246Cite as

Preparation of Proteins and Macromolecular Assemblies for Cryo-electron Microscopy

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2073))

Abstract

Cryo-electron microscopy has become popular as the penultimate step on the road to structure determination for many proteins and macromolecular assemblies. The process of obtaining high-resolution images of a purified biomolecular complex in an electron microscope often follows a long, and in many cases exhaustive screening process in which many iterative rounds of protein purification are employed and the sample preparation procedure progressively re-evaluated in order to improve the distribution of particles visualized under the electron microscope, and thus maximize the opportunity for high-resolution structure determination. Typically, negative stain electron microscopy is employed to obtain a preliminary assessment of the sample quality, followed by cryo-EM which first requires the identification of optimal vitrification conditions. The original methods for frozen-hydrated specimen preparation developed over 40 years ago still enjoy widespread use today, although recent developments have set the scene for a future where more systematic and high-throughput approaches to the preparation of vitrified biomolecular complexes may be routinely employed. Here we summarize current approaches and ongoing innovations for the preparation of frozen-hydrated single particle specimens for cryo-EM, highlighting some of the commonly encountered problems and approaches that may help overcome these.

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Authors and Affiliations

  1. School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, QLD, Australia

    Lou Brillault & Michael J. Landsberg

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  1. Lou Brillault
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  2. Michael J. Landsberg
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Correspondence to Michael J. Landsberg .

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  1. School of Biological Sciences, The MacDiarmid Institute for Advanced Materials and Nanotechnology, School of Chemical Sciences, University of Auckland, Auckland, New Zealand

    Juliet A. Gerrard

  2. School of Biological Sciences, Department of Chemical and Materials Engineering, The MacDiarmid Institute for Advanced Materials and Nanotechnology, University of Auckland, Auckland, New Zealand

    Laura J. Domigan

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Brillault, L., Landsberg, M.J. (2020). Preparation of Proteins and Macromolecular Assemblies for Cryo-electron Microscopy. In: Gerrard, J., Domigan, L. (eds) Protein Nanotechnology. Methods in Molecular Biology, vol 2073. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9869-2_13

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