title: A multicrystal diffraction data-collection approach for studying structural dynamics with millisecond temporal resolution.
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 forin situcrystallization 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.
authors.affiliation: 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.
authors.forename: Robin
authors.initials: R
authors.lastname: Schubert
authors.affiliation: University of Hamburg c/o DESY , Notkestrasse 85, 22603 Hamburg, Germany.
authors.forename: Svetlana
authors.initials: S
authors.lastname: Kapis
authors.affiliation: Center for Free Electron Laser Science c/o DESY , Notkestrasse 85, 22607 Hamburg, Germany.
authors.forename: Yannig
authors.initials: Y
authors.lastname: Gicquel
authors.affiliation: EMBL c/o DESY , Notkestrasse 85, 22603 Hamburg, Germany.
authors.forename: Gleb
authors.initials: G
authors.lastname: Bourenkov
authors.affiliation: EMBL c/o DESY , Notkestrasse 85, 22603 Hamburg, Germany.
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Schneider
authors.affiliation: Center for Free Electron Laser Science c/o DESY , Notkestrasse 85, 22607 Hamburg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Heymann
authors.affiliation: University of Hamburg c/o DESY , Notkestrasse 85, 22603 Hamburg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Betzel
authors.affiliation: 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.
authors.forename: Markus
authors.initials: M
authors.lastname: Perbandt
keywords: X-ray crystallography; fixed target; multicrystal data collection; protein structure; radiation damage; room temperature; structural biology; structure determination; synchrotron radiation; time-resolved crystallography