Helical crystallization of two example membrane proteins MsbA and the Ca(2+)-ATPase

Helical crystallization is a powerful tool for the moderate resolution structure determination of integral membrane proteins, where the insight gained often includes domain architecture and the disposition of α-helical segments. A necessary first step toward helical crystallization involves membrane protein reconstitution, which itself is a powerful technique for structure-function studies of integral membrane proteins. The correct insertion of a detergent-solubilized, purified membrane protein into lipid vesicles (proteoliposomes) can facilitate the functional characterization of the protein in a well-defined, chemically pure environment without interference from other membrane-associated components. In addition, the lipid-to-protein ratio can be controlled during reconstitution to generate a high concentration of a particular membrane protein in the proteoliposomes, which are then suitable for both functional assays and crystallization trials. Traditional approaches to two-dimensional crystallization for electron microscopy rely on dialysis methods for the simultaneous reconstitution and crystallization of a membrane protein [Kühlbrandt, W. (1992). Two-dimensional crystallization of membrane proteins. Q. Rev. Biophys.25, 1-49.], yet some systems allow these two steps to be experimentally separated and independently considered. Some examples of integral membrane proteins that have been reconstituted and crystallized in a helical lattice include cytochrome bc1 complex from bovine heart [Akiba, T., et al. (1996). Three-dimensional structure of bovine cytochrome bc(1) complex by electron cryomicroscopy and helical image reconstruction. Nat. Struct. Biol.3, 553-561.], Escherichia coli melibiose permease [Rigaud, J. L., et al. (1997). Bio-beads: An efficient strategy for two-dimensional crystallization of membrane proteins. J. Struct. Biol.118, 226-235.], a bacterial ATP-binding cassette transporter MsbA [Ward, A., et al. (2009). Nucleotide dependent packing differences in helical crystals of the ABC transporter MsbA. J. Struct. Biol.165, 169-175.], and the sarcoplasmic reticulum Ca(2+)-ATPase [Young, H. S., et al. (1997). How to make tubular crystals by reconstitution of detergent-solubilized Ca(2+)-ATPase. Biophys. J.72, 2545-2558.]. The reconstitution and helical crystallization of MsbA and Ca(2+)-ATPase will be the focus of this chapter.