Unit

UNIT 17.15 Overview of Electron Crystallography of Membrane Proteins: Crystallization and Screening Strategies Using Negative Stain Electron Microscopy

  1. Brent L. Nannenga1,
  2. Matthew G. Iadanza1,
  3. Breanna S. Vollmar1,
  4. Tamir Gonen1,2

Published Online: 1 APR 2013

DOI: 10.1002/0471140864.ps1715s72

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Nannenga, B. L., Iadanza, M. G., Vollmar, B. S. and Gonen, T. 2013. Overview of Electron Crystallography of Membrane Proteins: Crystallization and Screening Strategies Using Negative Stain Electron Microscopy. Current Protocols in Protein Science. 72:17.15:17.15.1–17.15.11.

Author Information

  1. 1

    Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia

  2. 2

    Corresponding author

Publication History

  1. Published Online: 1 APR 2013
  2. Published Print: APR 2013

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