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Sample Preparation for Electron Cryo-Microscopy of Macromolecular Machines

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Advanced Technologies for Protein Complex Production and Characterization

Part of the book series: Advances in Experimental Medicine and Biology ((AEMB,volume 1453))

Abstract

High-resolution structure determination by electron cryo-microscopy underwent a step change in recent years. This now allows study of challenging samples which previously were inaccessible for structure determination, including membrane proteins. These developments shift the focus in the field to the next bottlenecks which are high-quality sample preparations. While the amounts of sample required for cryo-EM are relatively small, sample quality is the key challenge. Sample quality is influenced by the stability of complexes which depends on buffer composition, inherent flexibility of the sample, and the method of solubilization from the membrane for membrane proteins. It further depends on the choice of sample support, grid pre-treatment and cryo-grid freezing protocol. Here, we discuss various widely applicable approaches to improve sample quality for structural analysis by cryo-EM.

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Acknowledgments

The authors would like to thank the members of the Berger and Schaffitzel team and Dr. Ufuk Borucu for critically reading the manuscript. CS acknowledges funding by the BBSRC (BB/P000940/1), the MRC (MR/P019471/1) and the Wellcome Trust (210701/Z/18/Z). AD acknowledges funding by the CEA DRF-Impulsion program (FIB-Bio grant).

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Authors and Affiliations

  1. Univ. Grenoble Alpes, CNRS, CEA, IRIG - Laboratoire de Chimie et Biologie des Métaux, Grenoble, France

    Aurélien Deniaud

  2. School of Biochemistry, University of Bristol, Bristol, UK

    Burak V. Kabasakal, Joshua C. Bufton & Christiane Schaffitzel

  3. Turkish Accelerator and Radiation Laboratory, Gölbaşı, Ankara, Türkiye

    Burak V. Kabasakal

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  1. Aurélien Deniaud
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  2. Burak V. Kabasakal
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  3. Joshua C. Bufton
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  4. Christiane Schaffitzel
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Correspondence to Christiane Schaffitzel .

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  1. Centro de Investigaciones Biologicas Margarita Salas (CIB-CSIC), Madrid, Spain

    M. Cristina Vega

  2. Abvance Biotech SL, Madrid, Spain

    Francisco J. Fernández

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Deniaud, A., Kabasakal, B.V., Bufton, J.C., Schaffitzel, C. (2024). Sample Preparation for Electron Cryo-Microscopy of Macromolecular Machines. In: Vega, M.C., Fernández, F.J. (eds) Advanced Technologies for Protein Complex Production and Characterization. Advances in Experimental Medicine and Biology, vol 1453. Springer, Cham. https://doi.org/10.1007/978-3-031-52193-5_12

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