Research Article: Outrunning free radicals in room-temperature macromolecular crystallography

Date Published: July 01, 2012

Publisher: International Union of Crystallography

Author(s): Robin L. Owen, Danny Axford, Joanne E. Nettleship, Raymond J. Owens, James I. Robinson, Ann W. Morgan, Andrew S. Doré, Guillaume Lebon, Christopher G. Tate, Elizabeth E. Fry, Jingshan Ren, David I. Stuart, Gwyndaf Evans.


A systematic increase in lifetime is observed in room-temperature protein and virus crystals through the use of reduced exposure times and a fast detector.

Partial Text

The cryocooling of crystals in macromolecular crystallography (MX) greatly increases their lifetime in the X-ray beam, and for this reason the vast majority of synchrotron-based MX is performed using crystals held at 100 K in an open-flow nitrogen cryostat (Garman & Owen, 2007 ▸). Despite the advantages provided by cryocooling, there is still considerable interest in carrying out room-temperature (RT) crystallo­graphy at synchrotron sources, and this has been reflected by the development of dedicated sample environments at several facilities (Jacquamet et al., 2004 ▸; Bingel-Erlenmeyer et al., 2011 ▸; Axford et al., 2012 ▸). The reasons for this interest are twofold. Firstly, screening of crystals in situ removes the invasive and potentially destructive step of crystal mounting and eliminates confounding factors such as cryoprotection and crystal handling when establishing optimal crystallization conditions. Secondly, some macromolecules, in particular viruses, prove to be difficult or indeed impossible to successfully cryocool, precluding data collection at 100 K. Additional motivation for collecting data at room temperature comes from recent work suggesting that data collection at 100 K can hide conformational diversity (Fraser et al., 2011 ▸). In both cases radiation damage becomes a limiting factor during data collection and it is often impossible to collect a complete data set from a single crystal, or indeed a small number of crystals.

The high doses required to record useful diffraction from the samples used in this study resulted in extremely rapid crystal decay. Crystal lifetimes can therefore only be determined over a small number of images (<25), resulting in some apparent variation in lifetime between crystals subjected to the same incident flux. To address this, and in order that overall trends in lifetime as a function of dose rate can be determined without crystal-to-crystal variation being a dominant factor, a large number of crystals have been used in this study (>110). All crystals exhibited an exponential decay in intensity and an exponential function with an R value greater than 0.9 could be fitted to the observed decay. It is important to note that the ability to record only a small number of images does not preclude structure solution: the structures of FcγRIIIa and BEV 2 have both been solved using RT data recorded in situ at I24 (Axford et al., 2012 ▸).

This study provides clear evidence for a dose-rate effect in RT macromolecular crystallography when using the full flux of an undulator beamline at a third-generation synchrotron in conjunction with a fast-readout detector. This study extends previous work on RT data collection and radiation damage (Cherezov et al., 2002 ▸; Southworth-Davies & Garman, 2007 ▸; Barker et al., 2009 ▸; Rajendran et al., 2011 ▸; Warkentin et al., 2012 ▸). The FcγRIIIa, BEV 2 and A2AAR crystals used in this study were subjected to maximum dose rates of 689, 886 and 995 kGy s−1, which are comparable to the maximum previously reported (Warkentin et al., 2012 ▸). In addition, the fast-readout detector enabled 25 fps to be collected. This is almost two orders of magnitude beyond previous studies with CCDs and image plates. At exposure times of less than 60 ms (>16 fps) lifetimes increase as a function of dose rate. We also found that the crystal lifetime reduced by a factor of two when there was a pause between frames within a data set when, for example, the X-ray detector is read out, in contrast to previous findings (Warkentin et al., 2012 ▸). Despite differences in experiment design between this and other studies, it is interesting to note the similarity in the crystal lifetimes observed. RT lifetimes are of a similar magnitude, i.e. ∼0.2 MGy, and are approximately two orders of magnitude less than lifetimes at 100 K, suggesting that the processes that dominate room-temperature X-ray-induced damage are essentially complete on timescales shorter than even those studied here.

The data presented here provide clear evidence for a dose-rate effect in RT crystallography when using the full flux of an undulator beamline. The effect becomes significant at exposure times less than 60 ms (frame rates of >16 Hz), with lifetimes increasing as a function of dose rate. Also apparent is a reduced crystal lifetime when there is a pause between frames within a data set when, for example, the X-ray detector is read out. Both of these observations can be explained by considering a three-part model for radiation damage at room temperature. On slow timescales (>1 s) radical diffusion and quenching within the solvent occur, while on fast timescales (<60 ms) radical diffusion, quenching and recombination are relevant. Differences in free-radical formation and propagation at RT, the latter of which is quite different between RT and 100 K, account for the absence of a significant dose-rate effect in cryocrystallography. These observations suggest that more intense beams and faster detectors might render RT data collection a generally attractive strategy for the collection of macromolecular crystallography data.   Source: