Header

UZH-Logo

Maintenance Infos

Cryo-electron microscopy of membrane proteins


Goldie, Kenneth N; Abeyrathne, Priyanka; Kebbel, Fabian; Chami, Mohamed; Ringler, Philippe; Stahlberg, Henning (2014). Cryo-electron microscopy of membrane proteins. Methods in Molecular Biology, 1117:325-41.

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.

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.

Statistics

Citations

15 citations in Web of Science®
8 citations in Scopus®
Google Scholar™

Altmetrics

Downloads

264 downloads since deposited on 10 Jul 2014
84 downloads since 12 months
Detailed statistics

Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:Special Collections > SystemsX.ch
Special Collections > SystemsX.ch > Research, Technology and Development Projects > CINA
Special Collections > SystemsX.ch > Research, Technology and Development Projects
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2014
Deposited On:10 Jul 2014 13:35
Last Modified:05 Apr 2016 17:57
Publisher:Springer
ISSN:1064-3745
Publisher DOI:https://doi.org/10.1007/978-1-62703-776-1_15
PubMed ID:24357370

Download

Preview Icon on Download
Preview
Filetype: PDF
Size: 1MB
View at publisher