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Radiation damage in room-temperature data acquisition with the PILATUS 6M pixel detector.

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.

SUBMITTER: Rajendran C

PROVIDER: S-EPMC3133521 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Radiation damage in room-temperature data acquisition with the PILATUS 6M pixel detector.

Rajendran Chitra C Dworkowski Florian S N FS Wang Meitian M Schulze-Briese Clemens C

Journal of synchrotron radiation 20110409 Pt 3

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 analy ...[more]

PMID: 21525639

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Project description:Protein crystallography data collection at synchrotrons is routinely carried out at cryogenic temperatures to mitigate radiation damage. Although damage still takes place at 100?K and below, the immobilization of free radicals increases the lifetime of the crystals by approximately 100-fold. Recent studies have shown that flash-cooling decreases the heterogeneity of the conformational ensemble and can hide important functional mechanisms from observation. These discoveries have motivated increasing numbers of experiments to be carried out at room temperature. However, the trade-offs between increased risk of radiation damage and increased observation of alternative conformations at room temperature relative to cryogenic temperature have not been examined. A considerable amount of effort has previously been spent studying radiation damage at cryo-temperatures, but the relevance of these studies to room temperature diffraction is not well understood. Here, the effects of radiation damage on the conformational landscapes of three different proteins (T. danielli thaumatin, hen egg-white lysozyme and human cyclophilin A) at room (278?K) and cryogenic (100?K) temperatures are investigated. Increasingly damaged datasets were collected at each temperature, up to a maximum dose of the order of 107?Gy at 100?K and 105?Gy at 278?K. Although it was not possible to discern a clear trend between damage and multiple conformations at either temperature, it was observed that disorder, monitored by B-factor-dependent crystallographic order parameters, increased with higher absorbed dose for the three proteins at 100?K. At 278?K, however, the total increase in this disorder was only statistically significant for thaumatin. A correlation between specific radiation damage affecting side chains and the amount of disorder was not observed. This analysis suggests that elevated conformational heterogeneity in crystal structures at room temperature is observed despite radiation damage, and not as a result thereof.

Dataset Information

Radiation damage in room-temperature data acquisition with the PILATUS 6M pixel detector.

Publications

Radiation damage in room-temperature data acquisition with the PILATUS 6M pixel detector.

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