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.