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

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.

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