Overview of Electron Crystallography of Membrane Proteins: Crystallization and Screening Strategies Using Negative Stain Electron Microscopy

Brent L. Nannenga1, Matthew G. Iadanza1, Breanna S. Vollmar1, Tamir Gonen2

1 Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, 2 Corresponding author
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 17.15
DOI:  10.1002/0471140864.ps1715s72
Online Posting Date:  April, 2013
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Abstract

Electron cryomicroscopy, or cryoEM, is an emerging technique for studying the three‐dimensional structures of proteins and large macromolecular machines. Electron crystallography is a branch of cryoEM in which structures of proteins can be studied at resolutions that rival those achieved by X‐ray crystallography. Electron crystallography employs two‐dimensional crystals of a membrane protein embedded within a lipid bilayer. The key to a successful electron crystallographic experiment is the crystallization, or reconstitution, of the protein of interest. This unit describes ways in which protein can be expressed, purified, and reconstituted into well‐ordered two‐dimensional crystals. A protocol is also provided for negative stain electron microscopy as a tool for screening crystallization trials. When large and well‐ordered crystals are obtained, the structures of both protein and its surrounding membrane can be determined to atomic resolution. Curr. Protoc. Protein Sci. 72:17.15.1‐17.15.11. © 2013 by John Wiley & Sons, Inc.

Keywords: electron crystallography; membrane proteins; negative stain electron microscopy; protein purification; protein solubilization

     
 
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Table of Contents

  • Introduction
  • Large‐Scale Production of Membrane Proteins
  • Membrane Protein Solubilization
  • Membrane Protein Purification
  • The Growth of 2D Crystals—Membrane Protein Reconstitution
  • Negative Staining EM as a Tool for Screening 2D Crystals
  • Conclusions
  • Acknowledgements
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

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Literature Cited

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