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Cryo-electron microscopy of membrane proteins.

Type of publication Peer-reviewed
Publikationsform Original article (peer-reviewed)
Author Goldie Kenneth N, Abeyrathne Priyanka, Kebbel Fabian, Chami Mohamed, Ringler Philippe, Stahlberg Henning,
Project Electron Microscopy of Membrane Proteins
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Original article (peer-reviewed)

Journal Methods in molecular biology (Clifton, N.J.)
Volume (Issue) 1117
Page(s) 325 - 41
Title of proceedings Methods in molecular biology (Clifton, N.J.)
DOI 10.1007/978-1-62703-776-1_15

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.
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