Radiation Damage in Macromolecular Crystallography

Abstract

Radiation damage inflicted on macromolecular crystals during X-ray diffraction experiments remains a limiting factor for structure solution, even when samples are cooled to cryotemperatures (~100 K). Efforts to establish mitigation strategies are ongoing and various approaches, summarized below, have been investigated over the last 15 years, resulting in a deeper understanding of the physical and chemical factors affecting damage rates. The recent advent of X-ray free electron lasers permits "diffraction-before-destruction" by providing highly brilliant and short (a few tens of fs) X-ray pulses. New fourth generation synchrotron sources now coming on line with higher X-ray flux densities than those available from third generation synchrotrons will bring the issue of radiation damage once more to the fore for structural biologists.

Dose-resolved serial synchrotron and XFEL structures of radiation-sensitive metalloproteins

“…Fundamentally, the structure obtained should be representative of the native state of the protein. However, macromolecular crystallography is typically carried out at cryogenic temperatures (100 K) to minimize radiation-damage-induced structural perturbation (Garman & Weik, 2017;Holton, 2009). There is an increasing recognition of the importance of determining structures at ambient or 'room' temperature so as to be more representative of the structures and dynamics adopted by proteins in vivo at physiological temperature (Keedy et al, 2018(Keedy et al, , 2015Fischer et al, 2015;Weik & Colletier, 2010).…”

Radiation chemists look at damage in redox proteins induced by X‐rays

“…A particularly comprehensive recent review is by Garman and Weik. 1 Following is a short summary of the key conclusions of these studies relevant to the present review.…”

“…9 Additionally, Compton scattering, involving loss of a valence electron and an X-ray photon of lower energy, contributes about 10% to the absorption events. 1 The free radicals produced (vide infra) can the protein or with other molecules in the crystals' that thus may act as radical scavengers, or recombine to form excited states. 9 While the free radicals would be limited in diffusion, electrons (and holes) can migrate by tunneling or hopping mechanisms, even at the low temperature commonly used in structural studies.…”

“…Further electron transfer to the Cu(II) site leads to the final lowest energy situation. 1 Disulfide modification is not the result of the direct photoionization of S, as this would lead to production of a cation radical and would also cause damage at methionine as well as at cystine, which is not commonly observed. 46 The Cu(II) ion, like sulfur, has a higher radiation absorption cross section than peptide atoms and is also especially susceptible to direct ionization.…”

“…Awareness of the potential radiation damage caused to macromolecules, predominantly proteins, during X-ray exposure has markedly increased with the mounting usage of the intense radiation produced by synchrotrons. 1 This review cannot end without mention of the promise held by yet higher intensity radiation sources from X-ray free-electron lasers. 77,78 These sources present one solution to some of the problems presented by synchrotron radiation.…”