title: Cryo-electron microscopy of membrane proteins.
abstract: Electron crystallography is used to study membrane proteins in the form of planar, two-dimensional (2D) crystals, or other crystalline arrays such as tubular crystals. This method has been used to determine the atomic resolution structures of bacteriorhodopsin, tubulin, aquaporins, and several other membrane proteins. In addition, a large number of membrane protein structures were studied at a slightly lower resolution, whereby at least secondary structure motifs could be identified.In order to conserve the structural details of delicate crystalline arrays, cryo-electron microscopy (cryo-EM) allows imaging and/or electron diffraction of membrane proteins in their close-to-native state within a lipid bilayer membrane.To achieve ultimate high-resolution structural information of 2D crystals, meticulous sample preparation for electron crystallography is of outmost importance. Beam-induced specimen drift and lack of specimen flatness can severely affect the attainable resolution of images for tilted samples. Sample preparations that sandwich the 2D crystals between symmetrical carbon films reduce the beam-induced specimen drift, and the flatness of the preparations can be optimized by the choice of the grid material and the preparation protocol.Data collection in the cryo-electron microscope using either the imaging or the electron diffraction mode has to be performed applying low-dose procedures. Spot-scanning further reduces the effects of beam-induced drift. Data collection using automated acquisition schemes, along with improved and user-friendlier data processing software, is increasingly being used and is likely to bring the technique to a wider user base.
authors.affiliation: Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University Basel, Basel, Switzerland.
authors.forename: Kenneth N
authors.initials: KN
authors.lastname: Goldie
authors.forename: Priyanka
authors.initials: P
authors.lastname: Abeyrathne
authors.forename: Fabian
authors.initials: F
authors.lastname: Kebbel
authors.forename: Mohamed
authors.initials: M
authors.lastname: Chami
authors.forename: Philippe
authors.initials: P
authors.lastname: Ringler
authors.forename: Henning
authors.initials: H
authors.lastname: Stahlberg
mesh_terms: D020285:Cryoelectron Microscopy / Q000379:methods*; D003461:Crystallography / Q000379:methods; D016591:Histocytological Preparation Techniques; D007091:Image Processing, Computer-Assisted; D008565:Membrane Proteins / Q000737:chemistry*
chemical_list: D008565:Membrane Proteins