Global radiation damage: temperature dependence, time dependence and how to outrun it

Abstract

A series of studies that provide a consistent and illuminating picture of global radiation damage to protein crystals, especially at temperatures above ∼200 K, are described. The radiation sensitivity shows a transition near 200 K, above which it appears to be limited by solvent-coupled diffusive processes. Consistent with this interpretation, a component of global damage proceeds on timescales of several minutes at 180 K, decreasing to seconds near room temperature. As a result, data collection times of order 1 s allow up to half of global damage to be outrun at 260 K. Much larger damage reductions near room temperature should be feasible using larger dose rates delivered using microfocused beams, enabling a significant expansion of structural studies of proteins under more nearly native conditions.

Analysis of global and site-specific radiation damage in cryo-EM

“…The D50 value of 2.2 e -Å -2 calculated from reflections of proteinase K in the 21.0-1.7 Å interval is consistent with past measurements using electron diffraction from two-dimensional crystals (Stark et al, 1996) as well as three-dimensional crystals Jeng & Chiu, 1984;Baker et al, 2010). When D50 was calculated from the reflections in the 14.0-1.7 Å interval of the much smaller hepta-petide, its value was 2.0 e -Å -2 and 2.2 e -Å -2 for high and low exposures, respectively, very close to the value obtained from proteinase K. In line with previous studies in synchrotron X-ray crystallography at comparable flux densities and temperatures, we do not see any effects from the dose rate on the observed global damage (Holton, 2009;Warkentin et al, 2013).…”

Spatial distribution of radiation damage to crystalline proteins at 25–300 K

“…The B-factor-derived dose estimates given above should thus be considered accurate to within 50%. However, unlike in our quantitative studies of global radiation damage (Kmetko et al, 2006(Kmetko et al, , 2011Warkentin & Thorne, 2010;Warkentin et al, 2011Warkentin et al, , 2012, none of the present conclusions rely on the precise global damage or dose estimates and are robust against sample-to-sample variability of global sensitivity.…”

“…As has been previously discussed, global sensitivity estimates exhibit significant ($50%) sample-to-sample variability (Meents et al, 2007(Meents et al, , 2010Warkentin & Thorne, 2010;Warkentin et al, 2012). The B-factor-derived dose estimates given above should thus be considered accurate to within 50%.…”

Radiation Damage in Macromolecular Crystallography—An Experimentalist’s View

“…Therefore, the rise in serial crystallographic techniques at synchrotrons and X-ray free-electron lasers (XFEL) has made room temperature measurements more popular and accessible again [40,41]. Nevertheless, cryocooling the crystal usually increases the crystal lifetime in X-ray measurements up to 100 times by reducing the mobility of most of the radicals formed during X-ray irradiation [19,42].…”