Outrunning free radicals in room-temperature macromolecular crystallography

Owen, R.L.; Axford, Danny; Nettleship, Joanne E.; Owens, Raymond J.; Robinson, Jim; Morgan, Ann W; Dore, A.S.; Lebon, Guillaume; Tate, Christopher G.; Fry, Elizabeth E.; Ren, Jingshan; Stuart, David I.; Evans, Gwyndaf

doi:10.1107/s0907444912012553

Cited by 90 publications

(86 citation statements)

References 48 publications

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“…Previous studies have reported inconsistent results concerning the dependence of the acidic residue decarboxylation rate on solvent accessibility (Weik et al, 2000;Fioravanti et al, 2007;Gerstel et al, 2015). The prevalence of radical attack from solvent channels surrounding the protein in the crystal is a questionable cause, considering previous observations indicating that the strongly oxidizing hydroxyl radical is immobile at 100 K (Allan et al, 2013;Owen et al, 2012). Furthermore, the suggested electron hole-trapping mechanism which induces decarboxylation within proteins at 100 K has no clear mechanistic dependence on the solventaccessible area of each carboxyl group.…”

Section: Discussionmentioning

confidence: 97%

RNA protects a nucleoprotein complex against radiation damage

Bury

McGeehan

Antson

et al. 2016

Acta Cryst Sect D Struct Biol

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Radiation damage during macromolecular X-ray crystallographic data collection is still the main impediment for many macromolecular structure determinations. Even when an eventual model results from the crystallographic pipeline, the manifestations of radiation-induced structural and conformation changes, the so-called specific damage, within crystalline macromolecules can lead to false interpretations of biological mechanisms. Although this has been well characterized within protein crystals, far less is known about specific damage effects within the larger class of nucleoprotein complexes. Here, a methodology has been developed whereby per-atom density changes could be quantified with increasing dose over a wide (1.3-25.0 MGy) range and at higher resolution (1.98 Å ) than the previous systematic specific damage study on a protein-DNA complex. Specific damage manifestations were determined within the large trp RNA-binding attenuation protein (TRAP) bound to a singlestranded RNA that forms a belt around the protein. Over a large dose range, the RNA was found to be far less susceptible to radiation-induced chemical changes than the protein. The availability of two TRAP molecules in the asymmetric unit, of which only one contained bound RNA, allowed a controlled investigation into the exact role of RNA binding in protein specific damage susceptibility. The 11-fold symmetry within each TRAP ring permitted statistically significant analysis of the Glu and Asp damage patterns, with RNA binding unexpectedly being observed to protect these otherwise highly sensitive residues within the 11 RNA-binding pockets distributed around the outside of the protein molecule. Additionally, the method enabled a quantification of the reduction in radiationinduced Lys and Phe disordering upon RNA binding directly from the electron density. IntroductionWith the wide use of high-flux third-generation synchrotron sources, radiation damage (RD) has once again become a dominant reason for the failure of structure determination using macromolecular crystallography (MX) in experiments conducted both at room temperature and under cryocooled conditions (100 K). Significant progress has been made in recent years in understanding the inevitable manifestations of X-ray-induced RD within protein crystals, and there is now a body of literature on possible strategies to mitigate the effects of RD (e.g. Zeldin, Brockhauser et al., 2013;Bourenkov & Popov, 2010). However, there is still no general consensus within the field on how to minimize RD during MX data collection, and debates on the dependence of RD progression on incident X-ray energy (Shimizu et al., 2007; Liebschner et ISSN 2059-7983 al., 2015) and the efficacy of radical scavengers (Allan et al., 2013) have yet to be resolved.RD manifests in two forms. Global radiation damage is observed within reciprocal space as the overall decay of the summed intensity of reflections detected within the diffraction pattern as dose increases (Garman, 2010;Murray & Garman, 2002). Dose is defined as the a...

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Section: Discussionmentioning

confidence: 97%

RNA protects a nucleoprotein complex against radiation damage

Bury

McGeehan

Antson

et al. 2016

Acta Cryst Sect D Struct Biol

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“…However, the microspectrophotometry data recorded from 1.5 to 270 kGy s~^ showed no signs of any systematic dose-rate dependence. Similarly, crystallographically the dose rate for cryocooled samples (unlike the case for room-temperature studies; Owen et al, 2012) has no clear effect at the macroscopic level, but it has been shown to be a factor in specific radiation damage with increased dose rate, resulting in small but measurable increased damage at radiation-sensitive sites (Se and S atoms; Leiros et al, 2006). Leiros et al (2006) studied maltooligosyltrehalose trehalohydrolase and trypsin with tenfold and 24-fold dose-rate differences, respectively.…”

Section: Residue Solvent Accessibility (A )mentioning

confidence: 96%

Insights into the mechanism of X-ray-induced disulfide-bond cleavage in lysozyme crystals based on EPR, optical absorption and X-ray diffraction studies

Sutton

Black

Mercer

et al. 2013

Acta Crystallogr D Biol Crystallogr

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“…Injectorbased serial crystallography pushes the principle to the extreme by exposing each crystal to X-rays only once, while providing a continuously refreshed supply of thousands of unexposed crystals. In contrast to the B0.7 MGy deposited per bR crystal in our previous synchrotron experiment 30 , each free-electron laser X-ray pulse subjected a crystal to a dose of 10 MGy, far above the suggested limit of under 1 MGy at room temperature 36,37 . Nevertheless, at the used pulse energy (B62 mJ after attenuation) and pulse duration of 75 fs (B6.57e 10 photons per pulse), the global radiation damage is not to be expected based on studies with the model system lysozyme 4,38 .…”

Section: Resultsmentioning

confidence: 81%