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Methods Enzymol. 2010;481:147-64. doi: 10.1016/S0076-6879(10)81007-9.

Liposomes on a streptavidin crystal: a system to study membrane proteins by cryo-EM.

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1
Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut, USA.

Abstract

In this chapter, we describe the preparation of cryo-EM specimens for random spherically constrained (RSC) single-particle reconstruction of membrane proteins. The specimen consists of liposomes into which the purified membrane protein is reconstituted at low density. The substrate is a 2D streptavidin crystal, which serves as an affinity surface that tethers the liposomes, which are doped with biotinylated lipids; the crystal can also serve as an image-quality and image-calibration reference. After subtraction of the crystal and lipid membrane contributions to the image, the remaining particle images can be used for 3D reconstruction.

PMID:
20887857
PMCID:
PMC3903115
DOI:
10.1016/S0076-6879(10)81007-9
[Indexed for MEDLINE]
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