Protein structure determination by single-wavelength anomalous diffraction phasing of X-ray free-electron laser data.

Karol Nass,Anton Meinhart,Sabine Botha,Robert L Shoeman,Mengning Liang,Ilme Schlichting,Lutz Foucar,R Bruce Doak,Garth Williams,Andrew Aquila,Thomas R M Barends,Sebastien Boutet,Alexander Gorel,Jason Koglin

doi:10.1107/s2052252516002980

Abstract

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) offers unprecedented possibilities for macromolecular structure determination of systems that are prone to radiation damage. However, phasing XFEL data de novo is complicated by the inherent inaccuracy of SFX data, and only a few successful examples, mostly based on exceedingly strong anomalous or isomorphous difference signals, have been reported. Here, it is shown that SFX data from thaumatin microcrystals can be successfully phased using only the weak anomalous scattering from the endogenous S atoms. Moreover, a step-by-step investigation is presented of the particular problems of SAD phasing of SFX data, analysing data from a derivative with a strong anomalous signal as well as the weak signal from endogenous S atoms.

Highlights

X-ray crystallography is a highly successful method for the determination of three-dimensional structures of molecules
Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) offers unprecedented possibilities for macromolecular structure determination of systems that are prone to radiation damage
Lysozyme Gd-derivative single-wavelength anomalous diffraction (SAD) phasing and automatic model building and automated model-building tools implemented in auto

Summary

Introduction

X-ray crystallography is a highly successful method for the determination of three-dimensional structures of molecules. Examples to date include the structure determination of microcrystals grown in vivo (Redecke et al, 2013; Sawaya et al, 2014) and in lipidic cubic phase (Kang et al, 2015; Liu et al, 2013; Zhang et al, 2015) and of metalloproteins such as photosystem II (Suga et al, 2015), cytochrome c oxidase (Hirata et al, 2014) and peroxidase (Chreifi et al, 2016). All of these successes relied on prior phase

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Conclusion