Development of a dose-limiting data collection strategy for serial synchrotron rotation crystallography.

Kazuya Hasegawa,Toru Nakatsu,Kunio Hirata,Masaki Yamamoto,Nipawan Nuemket,Keitaro Yamashita,Tomohiro Murai,Takashi Kumasaka,Go Ueno,Hideo Ago

doi:10.1107/s1600577516016362

Abstract

Serial crystallography, in which single-shot diffraction images are collected, has great potential for protein microcrystallography. Although serial femtosecond crystallography (SFX) has been successfully demonstrated, limited beam time prevents its routine use. Inspired by SFX, serial synchrotron crystallography (SSX) has been investigated at synchrotron macromolecular crystallography beamlines. Unlike SFX, the longer exposure time of milliseconds to seconds commonly used in SSX causes radiation damage. However, in SSX, crystals can be rotated during the exposure, which can achieve efficient coverage of the reciprocal space. In this study, mercury single-wavelength anomalous diffraction (Hg-SAD) phasing of the luciferin regenerating enzyme (LRE) was performed using serial synchrotron rotation crystallography. The advantages of rotation and influence of dose on the data collected were evaluated. The results showed that sample rotation was effective for accurate data collection, and the optimum helical rotation step depended on multiple factors such as multiplicity and partiality of reflections, exposure time per rotation angle and the contribution from background scattering. For the LRE microcrystals, 0.25° was the best rotation step for the achievable resolution limit, whereas a rotation step larger than or equal to 1° was favorable for Hg-SAD phasing. Although an accumulated dose beyond 1.1 MGy caused specific damage at the Hg site, increases in resolution and anomalous signal were observed up to 3.4 MGy because of a higher signal-to-noise ratio.

Highlights

Macromolecular crystallography (MX) has contributed to the structure determination of more than 109000 proteins as of September 2016, but there are still many challenging and scientifically important targets yet to have their structure solved
Datasets Da-1.2 to Da-26 and Ds-1.1ith (i = 1, 2, . . . , 25), all of which were collected with a helical rotation step of 0.5, were processed using XDS with the parameters defined for Rs-0.5
We have demonstrated the advantages and limitations of SS-ROX by evaluating the anomalous signal and Hg-single-wavelength anomalous diffraction (SAD) phasing of luciferin regenerating enzyme (LRE)

Summary

Introduction

Macromolecular crystallography (MX) has contributed to the structure determination of more than 109000 proteins as of September 2016 (http://www.wwpdb.org), but there are still many challenging and scientifically important targets yet to have their structure solved. One of the difficulties in structure determination of these targets is obtaining large, well diffracting crystals. A microbeam with a photon flux density of 1010 to 1011 photons sÀ1 mmÀ2 increases the diffraction intensity and decreases the background scattering from the surrounding mother liquor or buffer. Great care is needed when using a microbeam because radiation damage causes diffraction data quality to deteriorate (Holton, 2009; Garman, 2010). Great care is needed when using a microbeam because radiation damage causes diffraction data quality to deteriorate (Holton, 2009; Garman, 2010). Henderson (1990)

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Results

Conclusion