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IUCrJ. 2016 Oct 26;3(Pt 6):393-401. eCollection 2016 Nov 1.

A multicrystal diffraction data-collection approach for studying structural dynamics with millisecond temporal resolution.

Author information

1
University of Hamburg c/o DESY, Notkestrasse 85, 22603 Hamburg, Germany; The Hamburg Center for Ultrafast Imaging c/o DESY, Luruper Chaussee 149, 22761 Hamburg, Germany.
2
University of Hamburg c/o DESY , Notkestrasse 85, 22603 Hamburg, Germany.
3
Center for Free Electron Laser Science c/o DESY , Notkestrasse 85, 22607 Hamburg, Germany.
4
EMBL c/o DESY , Notkestrasse 85, 22603 Hamburg, Germany.

Abstract

Many biochemical processes take place on timescales ranging from femto-seconds to seconds. Accordingly, any time-resolved experiment must be matched to the speed of the structural changes of interest. Therefore, the timescale of interest defines the requirements of the X-ray source, instrumentation and data-collection strategy. In this study, a minimalistic approach for in situ crystallization is presented that requires only a few microlitres of sample solution containing a few hundred crystals. It is demonstrated that complete diffraction data sets, merged from multiple crystals, can be recorded within only a few minutes of beamtime and allow high-resolution structural information of high quality to be obtained with a temporal resolution of 40 ms. Global and site-specific radiation damage can be avoided by limiting the maximal dose per crystal to 400 kGy. Moreover, analysis of the data collected at higher doses allows the time-resolved observation of site-specific radiation damage. Therefore, our approach is well suited to observe structural changes and possibly enzymatic reactions in the low-millisecond regime.

KEYWORDS:

X-ray crystallography; fixed target; multicrystal data collection; protein structure; radiation damage; room temperature; structural biology; structure determination; synchrotron radiation; time-resolved crystallography

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