Abstract
The first study of room-temperature macromolecular crystallography data acquisition with a silicon pixel detector is presented, where the data are collected in continuous sample rotation mode, with millisecond read-out time and no read-out noise. Several successive datasets were collected sequentially from single test crystals of thaumatin and insulin. The dose rate ranged between ∼ 1320 Gy s(-1) and ∼ 8420 Gy s(-1) with corresponding frame rates between 1.565 Hz and 12.5 Hz. The data were analysed for global radiation damage. A previously unreported negative dose-rate effect is observed in the indicators of global radiation damage, which showed an approximately 75% decrease in D(1/2) at sixfold higher dose rate. The integrated intensity decreases in an exponential manner. Sample heating that could give rise to the enhanced radiation sensitivity at higher dose rate is investigated by collecting data between crystal temperatures of 298 K and 353 K. UV-Vis spectroscopy is used to demonstrate that disulfide radicals and trapped electrons do not accumulate at high dose rates in continuous data collection.
Highlights
Radiation damage to biological crystals during synchrotron data collection is a major obstacle in macromolecular structure determination
The data collection statistics of insulin and thaumatin with different dose rates at room temperature (RT) are shown in Tables 5 and 6, respectively
Data collection statistics of insulin and thaumatin crystals at cryogenic temperature are reproduced in Tables 2 and 3, respectively (Muller, 2010)
Summary
Radiation damage to biological crystals during synchrotron data collection is a major obstacle in macromolecular structure determination. While cryogenic cooling significantly reduces the detrimental effects of ionizing radiation, damage is commonly observed in the X-ray data collected at the thirdgeneration synchrotron sources, even at cryogenic temperatures. The dose limit (20 MGy) at which the diffraction intensity is predicted to fall to at least half of its initial value at cryo-temperatures was derived by Henderson (1990) for cryo-EM and experimentally confirmed for X-ray protein crystallography by Owen et al (2006) to be 43 MGy, and has been investigated by other researchers (Teng & Moffat, 2000, 2002; Kmetko et al, 2006). Despite significant progress in rational methods for cryoprotection in macromolecular crystallography (Alcorn & Juers, 2010), there are frequently cases in which crystals become disordered or introduce internal lattice changes In the case of RT data collection the reported dose limits are almost two orders of magnitude lower (Blake & Phillips, 1962; Nave & Garman, 2005; Southworth-Davies et al, 2007; Warkentin & Thorne, 2010).
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