Abstract
Serial crystallography is having an increasing impact on structural biology. This emerging technique opens up new possibilities for studying protein structures at room temperature and investigating structural dynamics using time-resolved X-ray diffraction. A limitation of the method is the intrinsic need for large quantities of well ordered micrometre-sized crystals. Here, a method is presented to screen for conditions that produce microcrystals of membrane proteins in the lipidic cubic phase using a well-based crystallization approach. A key advantage over earlier approaches is that the progress of crystal formation can be easily monitored without interrupting the crystallization process. In addition, the protocol can be scaled up to efficiently produce large quantities of crystals for serial crystallography experiments. Using the well-based crystallization methodology, novel conditions for the growth of showers of microcrystals of three different membrane proteins have been developed. Diffraction data are also presented from the first user serial crystallography experiment performed at MAX IV Laboratory.
Highlights
Serial crystallography at X-ray free-electron lasers and synchrotronsSynchrotron-radiation sources have been the most influential facilities for macromolecular protein crystallography (Dauter et al, 2010), and cryo-crystallography using single crystals is still the dominant method for solving protein structures at atomic resolution
Taking advantage of the extremely intense ultrashort microfocused X-ray pulses provided by X-ray free-electron lasers (XFELs), serial femtosecond crystallography (SFX) was developed, in which single-shot diffraction patterns are collected from a continuous stream of micrometre-sized crystals at room temperature before they are vaporized (Neutze et al, 2000; Chapman et al, 2011)
Sensory rhodopsin II (SRII; molecular weight 25 kDa) from Natronomonas pharaonis was purified according to Hohenfeld et al (1999) with the following modifications: the detergent n-decyl- -d-maltopyranoside was exchanged for n-octyl- -dglucopyranoside ( -OG), and a gel-filtration chromatography step was added with buffer consisting of 150 mM NaCl, 25 mM sodium/potassium phosphate, 0.8%(w/v) -OG pH 5.1 after the Ni–NTA chromatography to increase the purity before crystallization
Summary
Synchrotron-radiation sources have been the most influential facilities for macromolecular protein crystallography (Dauter et al, 2010), and cryo-crystallography using single crystals is still the dominant method for solving protein structures at atomic resolution. An advantage over traditional cryo-crystallography is that the need to grow large crystals is circumvented, and SFX has been successfully used to solve the structures of a number of membrane proteins The method of serial crystallography has been extended to include serial synchrotron crystallography (SSX) This storage-ring-based approach takes advantage of the improved characteristics of the new generation of synchrotrons with microfocused X-ray beams and improved optics, as well as low-noise high-frame-rate detectors and sophisticated software suites (Nogly et al, 2015; Weinert et al, 2017). SSX allows data collection on a millisecond time scale and makes the method accessible to a significantly larger number of users
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