Origin and temperature dependence of radiation damage in biological samples at cryogenic temperatures

Abstract

Radiation damage is the major impediment for obtaining structural information from biological samples by using ionizing radiation such as x-rays or electrons. The knowledge of underlying processes especially at cryogenic temperatures is still fragmentary, and a consistent mechanism has not been found yet. By using a combination of single-crystal x-ray diffraction, small-angle scattering, and qualitative and quantitative radiolysis experiments, we show that hydrogen gas, formed inside the sample during irradiation, rather than intramolecular bond cleavage between non-hydrogen atoms, is mainly responsible for the loss of high-resolution information and contrast in diffraction experiments and microscopy. The experiments that are presented in this paper cover a temperature range between 5 and 160 K and reveal that the commonly used temperature in x-ray crystallography of 100 K is not optimal in terms of minimizing radiation damage and thereby increasing the structural information obtainable in a single experiment. At 50 K, specific radiation damage to disulfide bridges is reduced by a factor of 4 compared to 100 K, and samples can tolerate a factor of 2.6 and 3.9 higher dose, as judged by the increase of R free values of elastase and cubic insulin crystals, respectively.

Glass transition in thaumatin crystals revealed through temperature-dependent radiation-sensitivity measurements

“…Using the radiationdamage metric of Kmetko et al (2006), we examined 49 thaumatin crystals to determine the radiation-sensitivity at 11 temperatures from 300 to 100 K. Our results agree with previous studies at 300 and 100 K and indicate that most of the reduction in sensitivity on cooling from 300 to 100 K occurs above 200 K. These results are consistent with previous reports on the temperature-dependence of global damage (Teng & Moffat, 2002;Borek et al, 2007;Meents et al, 2010). We model radiation damage as a thermally activated process with a large barrier and a small barrier.…”

“…For example, Meents et al (2010) recently reported measurements of radiation damage to two proteins (insulin and elastase) at temperatures between 160 and 5 K. They conclude that hydrogen gas formed inside the sample during irradiation is mainly responsible for the loss of highresolution information and contrast in diffraction experiments and electron microscopy, dominating over all of the radiationdamage mechanisms for this temperature range discussed here and in the previous literature.…”

Perovskite-Inspired Photovoltaic Materials: Toward Best Practices in Materials Characterization and Calculations

“…Similar to synchrotron measurements on other halide materials, 77 and biological materials, 78 significant beam damage can occur when working with MAPbI 3 and other halide-based PIMs. It is therefore important to consider optimal conditions for measuring these new absorbers.…”

Temperature-dependent macromolecular X-ray crystallography

“…Only a small increase in crystal lifetime of about 25% has been reported for data collected at 15 K instead of 100 K as judged from global radiation-damage indicators (Chinte et al, 2007;Meents et al, 2007). With respect to that at 100 K, at 50 K specific damage to a disulfide bond has been reported to be reduced fourfold and global damage to be decreased by 50% (Meents et al, 2010). On the other hand, the photoreduction of metal centres, a specific radiation-damage effect (Yano et al, 2005), is reduced 30-fold at 40 K compared with 110 K as determined by X-ray absorption spectroscopy (Corbett et al, 2007).…”