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Specific radiation damage is a lesser concern at room temperature.

ABSTRACT: Carrying out macromolecular crystallography (MX) experiments at cryogenic temperatures significantly slows the rate of global radiation damage, thus facilitating the solution of high-resolution crystal structures of macromolecules. However, cryo-MX experiments suffer from the early onset of so-called specific radiation damage that affects certain amino-acid residues and, in particular, the active sites of many proteins. Here, a series of MX experiments are described which suggest that specific and global radiation damage are much less decoupled at room temperature than they are at cryogenic temperatures. The results reported here demonstrate the interest in reviving the practice of collecting MX diffraction data at room temperature and allow structural biologists to favourably envisage the development of time-resolved MX experiments at synchrotron sources.

SUBMITTER: Gotthard G

PROVIDER: S-EPMC6608634 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Specific radiation damage is a lesser concern at room temperature.

Gotthard Guillaume G Aumonier Sylvain S De Sanctis Daniele D Leonard Gordon G von Stetten David D Royant Antoine A

IUCrJ 20190612 Pt 4

Carrying out macromolecular crystallography (MX) experiments at cryogenic temperatures significantly slows the rate of global radiation damage, thus facilitating the solution of high-resolution crystal structures of macromolecules. However, cryo-MX experiments suffer from the early onset of so-called specific radiation damage that affects certain amino-acid residues and, in particular, the active sites of many proteins. Here, a series of MX experiments are described which suggest that specific a ...[more]

PMID: 31316810

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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.

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Specific radiation damage is a lesser concern at room temperature.

Publications

Specific radiation damage is a lesser concern at room temperature.

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