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Title: Conformational variation of proteins at room temperature is not dominated by radiation damage

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 10 7 Gy at 100 K and 10 5 Gy at 278 K. Although it wasmore » 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. Lastly, this analysis suggests that elevated conformational heterogeneity in crystal structures at room temperature is observed despite radiation damage, and not as a result thereof.« less
Authors:
 [1] ;  [1] ;  [2] ;  [2] ; ORCiD logo [2] ; ORCiD logo [1]
  1. SLAC National Accelerator Lab., Menlo Park, CA (United States)
  2. UCSF, San Francisco, CA (United States)
Publication Date:
Grant/Contract Number:
AC02-76SF00515
Type:
Accepted Manuscript
Journal Name:
Journal of Synchrotron Radiation (Online)
Additional Journal Information:
Journal Name: Journal of Synchrotron Radiation (Online); Journal Volume: 24; Journal Issue: 1; Journal ID: ISSN 1600-5775
Publisher:
International Union of Crystallography
Research Org:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22); USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); National Institutes of Health (NIH)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.; radiation damage; room temperature; cryo-temperature; conformational dynamics
OSTI Identifier:
1348844

Russi, Silvia, González, Ana, Kenner, Lillian R., Keedy, Daniel A., Fraser, James S., and van den Bedem, Henry. Conformational variation of proteins at room temperature is not dominated by radiation damage. United States: N. p., Web. doi:10.1107/S1600577516017343.
Russi, Silvia, González, Ana, Kenner, Lillian R., Keedy, Daniel A., Fraser, James S., & van den Bedem, Henry. Conformational variation of proteins at room temperature is not dominated by radiation damage. United States. doi:10.1107/S1600577516017343.
Russi, Silvia, González, Ana, Kenner, Lillian R., Keedy, Daniel A., Fraser, James S., and van den Bedem, Henry. 2017. "Conformational variation of proteins at room temperature is not dominated by radiation damage". United States. doi:10.1107/S1600577516017343. https://www.osti.gov/servlets/purl/1348844.
@article{osti_1348844,
title = {Conformational variation of proteins at room temperature is not dominated by radiation damage},
author = {Russi, Silvia and González, Ana and Kenner, Lillian R. and Keedy, Daniel A. and Fraser, James S. and van den Bedem, Henry},
abstractNote = {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. Lastly, this analysis suggests that elevated conformational heterogeneity in crystal structures at room temperature is observed despite radiation damage, and not as a result thereof.},
doi = {10.1107/S1600577516017343},
journal = {Journal of Synchrotron Radiation (Online)},
number = 1,
volume = 24,
place = {United States},
year = {2017},
month = {1}
}