title: Development of tools to automate quantitative analysis of radiation damage in SAXS experiments.
abstract: Biological small-angle X-ray scattering (SAXS) is an increasingly popular technique used to obtain nanoscale structural information on macromolecules in solution. However, radiation damage to the samples limits the amount of useful data that can be collected from a single sample. In contrast to the extensive analytical resources available for macromolecular crystallography (MX), there are relatively few tools to quantitate radiation damage for SAXS, some of which require a significant level of manual characterization, with the potential of leading to conflicting results from different studies. Here, computational tools have been developed to automate and standardize radiation damage analysis for SAXS data. RADDOSE-3D, a dose calculation software utility originally written for MX experiments, has been extended to account for the cylindrical geometry of the capillary tube, the liquid composition of the sample and the attenuation of the beam by the capillary material to allow doses to be calculated for many SAXS experiments. Furthermore, a library has been written to visualize and explore the pairwise similarity of frames. The calculated dose for the frame at which three subsequent frames are determined to be dissimilar is defined as the radiation damage onset threshold (RDOT). Analysis of RDOTs has been used to compare the efficacy of radioprotectant compounds to extend the useful lifetime of SAXS samples. Comparison of the RDOTs shows that, for radioprotectant compounds at 5 and 10 mM concentration, glycerol is the most effective compound. However, at 1 and 2 mM concentrations, dithiothreitol (DTT) appears to be most effective. Our newly developed visualization library contains methods that highlight the unusual radiation damage results given by SAXS data collected using higher concentrations of DTT: these observations should pave the way to the development of more sophisticated frame merging strategies.
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Brooks-Bartlett
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Batters
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.
authors.forename: Charles S
authors.initials: CS
authors.lastname: Bury
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.
authors.forename: Edward D
authors.initials: ED
authors.lastname: Lowe
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford OX3 7BN, UK.
authors.forename: Helen Mary
authors.initials: HM
authors.lastname: Ginn
authors.affiliation: European Molecular Biology Laboratory, Grenoble Outstation, 71 avenue des Martyrs, CS 90181, 38042 Grenoble, France.
authors.forename: Adam
authors.initials: A
authors.lastname: Round
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.
authors.forename: Elspeth F
authors.initials: EF
authors.lastname: Garman
mesh_terms: D006801:Humans; D046911:Macromolecular Substances / Q000737:chemistry*; D011506:Proteins; D053838:Scattering, Small Angle; D012984:Software; D014961:X-Ray Diffraction
chemical_list: D046911:Macromolecular Substances; D011506:Proteins
keywords: CorMap visualization; RADDOSE-3D; SAXS; radiation damage; radioprotectants