Liposomes on a streptavidin crystal: a system to study membrane proteins by cryo-EM.
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.
Wang
Liguo
L
Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut, USA.
Sigworth
Fred J
FJ
D020285:Cryoelectron Microscopy / Q000379:methods*; D007091:Image Processing, Computer-Assisted; D008081:Liposomes / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D019809:Streptavidin / Q000737:chemistry*
D008081:Liposomes; D008565:Membrane Proteins; D019809:Streptavidin