Abstract
A reliable and reproducible method to automatically characterize the radiation sensitivity of macromolecular crystals at the ESRF beamlines has been developed. This new approach uses the slope of the linear dependence of the overall isotropic B-factor with absorbed dose as the damage metric. The method has been implemented through an automated procedure using the EDNA on-line data analysis framework and the MxCuBE data collection control interface. The outcome of the procedure can be directly used to design an optimal data collection strategy. The results of tests carried out on a number of model and real-life crystal systems are presented.
Highlights
Radiation damage incurred during data collection in macromolecular crystallography (MX) limits the information that can be obtained from a single crystal
Consideration of radiation damage effects is critical for optimal data collection planning
Six crystal systems were selected for testing the method: thermolysin from Bacillus thermoproteolyticus (MuellerDieckmann et al, 2007); bovine pancreatic trypsin (Bartunik et al, 1989); a ten base pair oligonucleotide d(AGGGGCCCCT)2 A-DNA (Leal et al, 2009); Se-Met containing FAE, feruloyl esterase module of xylanase 10B from Clostridium thermocellum (Prates et al, 2001); RecR from Deinococcus radiodurans (Lee et al, 2004); and the 1-adrenergic G-protein coupled receptor (GPCR) from Meleagris gallopavo (Warne et al, 2008)
Summary
Radiation damage incurred during data collection in macromolecular crystallography (MX) limits the information that can be obtained from a single crystal. It occurs at any temperature and leads to a resolution-dependent reduction in diffraction intensity, changes in the unit-cell parameters and crystal mosaicity as well as slight rotations and translations of macromolecules in the lattice It induces specific chemical changes (e.g. disulphide bond breaks, decarboxylation of acidic residues, changes in the oxidation state of metal ions) which may prevent the structure solution or mislead biological interpretations. EDNA has been recently integrated in the ESRF beamline control interface, the MxCube software (Gabadinho et al, 2011), allowing for ‘one click’ sample characterization This feature fully characterizes the crystal sample and generates a data collection strategy that accounts for radiation damage. Using test crystals with well known radiation sensitivity, the procedure can be used at the beamlines to verify and calibrate flux and beam size
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