Charles S. Bury

Radiation damage to nucleoprotein complexes in macromolecular crystallography.

Quantitative X-ray induced radiation damage studies employing a model protein–DNA complex revealed a striking partition of damage sites. The DNA component was observed to be far more resistant to specific damage compared with the protein.

RNA protects a nucleoprotein complex against radiation damage

Systematic analysis of radiation damage within a protein–RNA complex over a large dose range (1.3–25 MGy) reveals significant differential susceptibility of RNA and protein. A new method of difference electron-density quantification is presented.

Development of tools to automate quantitative analysis of radiation damage in SAXS experiments.

Radiation damage analysis with experimental SAXS data allows for the quantitative comparison of the efficacy of various additive radioprotectant compounds. Relevant extensions to RADDOSE-3D and the creation of a new visualization library to enable this study are presented.

OH cleavage from tyrosine: debunking a myth

A systematic macromolecular crystallography investigation into the observed electron density loss around the –OH group of tyrosines, as a function of dose at 100 K, is reported. It is concluded that a probable explanation is aromatic ring disordering as opposed to –OH cleavage; occurrence of the latter mechanism is a misconception perpetuated in radiation damage literature, and is unsupported by any observations in radiation chemistry.

Estimate your dose: RADDOSE-3D: RADDOSE-3D

We present the current status of RADDOSE‐3D, a software tool allowing the estimation of the dose absorbed in a macromolecular crystallography diffraction experiment. The code allows a temporal and spatial dose contour map to be calculated for a crystal of any geometry and size as it is rotated in an X‐ray beam, and gives several summary dose values: among them diffraction weighted dose. This allows experimenters to plan data collections which will minimize radiation damage effects by spreading the absorbed dose more homogeneously, and thus to optimize the use of their crystals. It also allows quantitative comparisons between different radiation damage studies, giving a universal “x‐axis” against which to plot various metrics.

Objective classification of specific radiation damage in macromolecular X-ray crystallography

crystallography Charles S. Bury, John E. McGeehan, Ian Carmichael, Elspeth F. Garman Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, King Henry I Street, Portsmouth, Hampshire PO1 2DY, UK Notre Dame Radiation Laboratory, University of Notre Dame, Notre Dame, IN 46556, USA

Mechanism and X-ray damage: beware of artefacts in xylose isomerase

Radiation chemistry causes structural perturbations with significant impact to the accurate structural understanding of mechanism, especially in metalloproteins. Xylose isomerase (XI) is an example of this issue: it catalyzes the reversible interconversion of the aldoses D-xylose, D-ribose, and D-glucose to the corresponding ketoses. XI requires a divalent metal ion such as Mn2+, Mg2+ or Co2+ as a cofactor [1]. It is an important industrial enzyme; XI is used extensively in the food industry, for example in producing high-fructose corn syrup, and it also holds enormous potential for application in converting biomass into fuels and chemicals. Thus understanding its mechanism and thereby potentially improving its suitability for industrial applications is even more important. The reaction mechanism has been studied in increasingly detailed studies, many discussing the mobility of the catalytic metal ion. We show that this mobility can be driven by the Xrays used to reveal the structure, and that it may not be mechanistically critical.

Radiation damage in macromolecular crystallography: the current knowns and unknowns

Much progress has been made over the last 15 years in characterising radiation damage (RD) to macromolecular crystals during 100 K X-ray diffraction experiments [1], and to a lesser extent, for those irradiated at room temperature (RT). Despite a now extensive body of literature on various aspects of RD in MX and SAXS (e.g. Special RD Issues of the Journal of Synchrotron Radiation in Nov 2002, May 2005, Jan 2007, March 2009, Jan 2013, March 2015, and Jan 2017), and new tools being developed to better characterise the effects, there are still a number of unanswered questions in our understanding of RD phenomena and in our knowledge of the pertinent radiation chemistry. Since these effects can not only prevent structure solution, but can also compromise the biological interpretation of structures, an awareness of the issue and of RD-induced artefacts is important for all structural biologists. Systematic work on the topic has included attempts to identify suitable robust metrics for monitoring both global and specific damage, and establishing an appropriate quantity against which to plot these metrics: the most useful x-axis is absorbed dose rather than time, image number or photons per second. Dose cannot be directly measured but can be estimated (for example by the software program RADDOSE-3D [2]), from knowledge of the atomic contents and size of the crystal, and the X-ray beam properties (energy, flux, profile, size). This information allows the absorption coefficients to be calculated and the dose distributions for various experimental strategies to be compared, in order to optimise the use of the crystal volume and minimise inhomogeneous irradiation. In addition, comparisons can then more easily be made between experiments carried out by different researchers using a range of X-ray facilities. RD related knowns and unknowns will be summarised, and some new approaches to electron density loss quantitation will be reported. In addition, we are working to improve dose estimations in RADDOSE-3D by explicitly taking into account both fluorescent X-ray escape and photoelectron escape from microcrystals [3], thus reducing the absorbed dose. Our model will allow the relative dose tolerances of micro and macro crystals to be estimated, which has implications for planning serial synchrotron crystallography experiments.