Advances in in situ X-ray Crystallography of Membrane Proteins

Abstract

In the last decade, crystallisation of membrane proteins in lipidic cubic phases (LCP; in meso) boosted the numbers and resolution of membrane-protein structures. However, membrane-protein crystals are usually small and notoriously difficult to harvest from the highly viscous LCP. Recently, an in situ in meso approach has been introduced, in which crystals are not harvested and flash-frozen but placed in the X-ray beam within the mesophase at room temperature. In this study, we introduce novel in situ in meso plates that show significantly less background scattering, are easier to handle, and are variable with respect to the crystallisation volume. Moreover, we developed holders for either individual or up to four in situ wells at a time, which are easy and cheap to produce, easy to handle, reusable, and compatible with measurements at room temperature and under cryogenic conditions. Because the holders are attached to standard ALS-type goniometer bases, they allow for storage and shipping of entire wells (with typically several dozens of crystals) in Universal V1-Pucks under liquid nitrogen and for auto-mounting at synchrotrons. We validated the new setups using water-soluble hen egg lysozyme and the membrane protein bacteriorhodopsin. In conclusion, this study demonstrates the potential of combining in meso crystallisation with in situ diffraction and the possibility to store, ship, and measure crystals under cryogenic conditions for obtaining structural information on membrane proteins. 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.