Datensatz

Zusammenfassung

Publisher Summary: The high-resolution structure analysis of membrane proteins is still a formidable task. In addition, no recombinant eucaryotic membrane protein has as yet been amenable to 3D crystallization. Soluble fragments of membrane proteins have been overexpressed, purified, and examined by conventional X-ray crystallography. However, this approach does not allow examination of transmembrane domains, which are involved in signal transduction and transport across membranes. Solid state nuclear magnetic resonance (NMR) spectroscopy has successfully been used to discuss the transmembrane domains of membrane proteins. However, the protein must be examined in micelles and the molecular weight limit precludes examination of complex polytopic proteins. An alternative approach is to grow 2D crystals and use electron cryomicroscopy and image analysis to solve the structure. Currently, the structural characterization of the majority of membrane proteins by electron cryocrystallography is indeed a challenging endeavor because the proteins are often present in low abundance in their native membranes, and only a few hundred micrograms of purified recombinant protein may be available. This chapter provides a general outline for the steps involved in the structure analysis of membrane proteins by electron cryocrystallography. The chapter presents results on the analysis of two-dimensional crystals of gap junction channels, aquaporin channels, and histidine-tagged Fab molecules, to exemplify respectively the general methods of in situ 2D crystallization, in vitro 2D crystallization, and lipid monolayer crystallization. Also discussed are the preparation of frozen-hydrated specimens, performance of low-dose transmission electron cryomicroscopy, and image analysis of 2D crystals.