title: Overview of electron crystallography of membrane proteins: crystallization and screening strategies using negative stain electron microscopy.
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.
authors.affiliation: Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
authors.forename: Brent L
authors.initials: BL
authors.lastname: Nannenga
authors.forename: Matthew G
authors.initials: MG
authors.lastname: Iadanza
authors.forename: Breanna S
authors.initials: BS
authors.lastname: Vollmar
authors.forename: Tamir
authors.initials: T
authors.lastname: Gonen
mesh_terms: D020285:Cryoelectron Microscopy / Q000379:methods*; D003461:Crystallography / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry*; D008968:Molecular Conformation; D016624:Negative Staining / Q000379:methods*
chemical_list: D008565:Membrane Proteins
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