Abstract
De novo membrane protein structure determination is often limited by the availability of large crystals and the difficulties in obtaining accurate diffraction data for experimental phasing. Here we present a method that combines in situ serial crystallography with de novo phasing for fast, efficient membrane protein structure determination. The method enables systematic diffraction screening and rapid data collection from hundreds of microcrystals in in meso crystallization wells without the need for direct crystal harvesting. The requisite data quality for experimental phasing is achieved by accumulating diffraction signals from isomorphous crystals identified post-data collection. The method works in all experimental phasing scenarios and is particularly attractive with fragile, weakly diffracting microcrystals. The automated serial data collection approach can be readily adopted at most microfocus macromolecular crystallography beamlines.
Highlights
De novo membrane protein structure determination is often limited by the availability of large crystals and the difficulties in obtaining accurate diffraction data for experimental phasing
Recent advances in cryogenic electron microscopy and de novo prediction methods will undoubtedly contribute to providing much-needed new structures[3,4]
Instead of harvesting mesophase-containing individual or clusters of crystals from each well by the traditional loop-harvesting method, the entire IMISX well was removed from the plate, mounted on a pin and snap-cooled in liquid nitrogen (Methods, Supplementary Fig. 2)
Summary
De novo membrane protein structure determination is often limited by the availability of large crystals and the difficulties in obtaining accurate diffraction data for experimental phasing. Harvesting of the large numbers of crystals required for serial data collection is very time-consuming and challenging for crystals grown in meso This background serves to highlight the requirement for efficient and robust de novo phasing methods for use with membrane protein microcrystals. Responding to this critical need, we introduce here a fully automated method for collecting, selecting, and merging data from hundreds to thousands of in meso-grown microcrystals This approach boosts the collective phasing signal of many tiny crystals to a level where de novo phasing of highly challenging membrane protein targets becomes possible and fast and high-throughput. We refer to it as the in meso in situ serial crystallography—experimental phasing (IMISX-EP) method. We demonstrate the utility of the method with four real-life integral membrane proteins using all major anomalous phasing methods, and show that IMISX-EP provides a fast, efficient, and a direct means for de novo structure determination of membrane protein as microcrystals
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