Serial crystallography onin vivogrown microcrystals using synchrotron radiation

Cornelius Gati,Henry N Chapman,Dirk Rehders,Thomas R Schneider,Oleksandr Yefanov,Marco Klinge,Gleb Bourenkov,Christian Betzel,Francesco Stellato,Dominik Oberthür,Benjamin P Sommer,Stefan Mogk,Lars Redecke,Michael Duszenko

doi:10.1107/s2052252513033939

Abstract

Crystal structure determinations of biological macromolecules are limited by the availability of sufficiently sized crystals and by the fact that crystal quality deteriorates during data collection owing to radiation damage. Exploiting a micrometre-sized X-ray beam, high-precision diffractometry and shutterless data acquisition with a pixel-array detector, a strategy for collecting data from many micrometre-sized crystals presented to an X-ray beam in a vitrified suspension is demonstrated. By combining diffraction data from 80 Trypanosoma brucei procathepsin B crystals with an average volume of 9 µm(3), a complete data set to 3.0 Å resolution has been assembled. The data allowed the refinement of a structural model that is consistent with that previously obtained using free-electron laser radiation, providing mutual validation. Further improvements of the serial synchrotron crystallography technique and its combination with serial femtosecond crystallography are discussed that may allow the determination of high-resolution structures of micrometre-sized crystals.

Highlights

Macromolecular crystallography (MX) is a powerful method for obtaining structural information about biological macromolecules and their assemblies
For detailed comparison of the T. brucei procathepsin B structure solved in this study at 110 K using synchrotron radiation with that previously obtained at room temperature using the free-electron laser (FEL)-based SFX technique (PDB entry 4hwy; Redecke et al, 2013), electron-density maps were generated using the SFX data truncated at 3.0 Aresolution
In recent years, combining data from multiple crystals has enabled the determination of a number of important structures (Rasmussen et al, 2011; Siu et al, 2013; Li et al, 2013), despite the difficulties arising from systematic errors when data from multiple crystals are merged

Summary

Introduction

Macromolecular crystallography (MX) is a powerful method for obtaining structural information about biological macromolecules and their assemblies. Data are collected in a second step using the rotation method (Arndt & Wonacott, 1977) from positions where diffraction was detected The application of this method has been crucial in the structure determination of GPCRs, where the crystals are small (of the order of 25 Â 6 Â 4 mm3) and invisible to optical light because the lipidic mesophase in which the crystals grow turns opaque upon cryogenic cooling. Inspired by the SFX methodology and the new capabilities to process large data sets, as well as the successful structure determination of TbCatB in the pro-form using the SFX approach, we embarked on an experiment to determine the crystal structure of T. brucei procathepsin B using a suspension of in vivo grown microcrystals mounted in a standard nylon loop for crystallographic data collection on a microfocus synchrotron beamline. Synchrotron and FEL radiation sources provided a unique opportunity to validate the results obtained against each other, and we present a comparison of the crystallographic models

Sample preparation and data collection

Data processing and structure determination

Comparison of the structural TbCatB models

Discussions and conclusions