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2D Electron Crystallography of Membrane Protein Single-, Double-, and Multi-Layered Ordered Arrays

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cryoEM

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2215))

Abstract

The electron cryo-microscopy (cryo-EM) approach of 2D electron crystallography allows for structure determination of two-dimensional (2D) crystals of soluble and membrane proteins, employing identical principles and methods once 2D crystals are obtained. Two-dimensional crystallization trials of membrane proteins can result in multiple outcomes of ordered arrays, which may be suited for either 2D electron crystallography, helical analysis, or MicroED.

The membrane protein 2D crystals used for 2D electron crystallography are either single- or double-layered ordered proteoliposome vesicles or sheet-like membranes. We have developed a cryo-EM grid preparation approach, which allows for the analysis of stacked 2D crystals that are neither suitable for MicroED nor for directly applying 2D electron crystallography. This new grid preparation approach, the peel-blot, uses the capillary force generated by submicron filter paper and mechanical means for the separation of stacked 2D crystals into single-layered 2D crystals, for which standard 2D electron crystallography can then be employed. The preparation of 2D crystals, the peel-blot grid preparation, and the structure determination by 2D electron crystallography are described here.

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Acknowledgments

Part of this work was supported by NIH grant HL090630 (ISK) and an SREB Fellowship (KN).

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Authors and Affiliations

  1. Structural Biology, Genentech, San Francisco, CA, USA

    Matthew C. Johnson

  2. School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA, USA

    Yusuf M. Uddin, Kasahun Neselu & Ingeborg Schmidt-Krey

  3. School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, USA

    Ingeborg Schmidt-Krey

Authors
  1. Matthew C. Johnson
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  2. Yusuf M. Uddin
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  3. Kasahun Neselu
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  4. Ingeborg Schmidt-Krey
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Corresponding author

Correspondence to Ingeborg Schmidt-Krey .

Editor information

Editors and Affiliations

  1. Howard Hughes Medical Institute Departments of Biological Chemistry and Physiology, University of California, Los Angeles, Los Angeles, CA, USA

    Tamir Gonen

  2. Chemical Engineering, School for Engineering of Matter, Transport and Energy; Center for Applied Structural Discovery Biodesign Institute, Arizona State University, Tempe, AZ, USA

    Brent L. Nannenga

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Johnson, M.C., Uddin, Y.M., Neselu, K., Schmidt-Krey, I. (2021). 2D Electron Crystallography of Membrane Protein Single-, Double-, and Multi-Layered Ordered Arrays. In: Gonen, T., Nannenga, B.L. (eds) cryoEM. Methods in Molecular Biology, vol 2215. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0966-8_10

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  • DOI: https://doi.org/10.1007/978-1-0716-0966-8_10

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0965-1

  • Online ISBN: 978-1-0716-0966-8

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