Advances in membrane-protein crystallization: From detergent-free crystallization to in situ approaches

We report the detergent-free crystallization of a membrane protein that has been in a lipid bilayer at every stage since its production in the cell. To do so, we combined styrene maleic acid (SMA) nanodiscs with crystallization in lipidic cubic phases (LCP). The 2.0-Å structure of an α-helical 7-transmembrane microbial rhodopsin thus obtained is of excellent quality and virtually identical to the 2.2-Å LCP structure obtained from a traditional detergent-based approach. This study is of fundamental interest, because it may allow to get structural insight into proteins, which so far have not been amenable to solubilization in detergents.

Membrane-protein structure determination is also impaired by the difficulties associated with harvesting small membrane-protein crystals from the highly viscous LCP. Recently, an in situ approach has been introduced, in which crystals are not harvested and flash-frozen but placed in the X-ray beam within the LCP. We introduce novel in situ plates that show significantly less background scattering, are cheaper, and easier to handle. Moreover, we developed a variety of holders, which are suited for measurements at room temperature and/or under cryogenic conditions. Some allow for storage and shipping of entire wells (with typically several dozens of crystals) in liquid nitrogen and are compatible with auto-mounting at synchrotrons, while others are perfect for rapid and automated screening of different crystallization conditions at room temperature. We validated the new setups using water-soluble hen egg lysozyme and myoglobin, as well as the membrane protein bacteriorhodopsin. In conjunction with the current developments at synchrotrons like smaller beams, faster detectors, and software for multi-crystal strategies, this approach promises high-resolution structural studies of membrane proteins to become faster and more routine.