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

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.

Doses for X-ray and electron diffraction: New features in RADDOSE-3D including intensity decay models.

New features in the dose estimation program RADDOSE-3D are summarised. They include the facility to enter a diffraction intensity decay model which modifies the "Diffraction Weighted Dose" output from a "Fluence Weighted Dose" to a "Diffraction-Decay Weighted Dose", a description of RADDOSE-ED for use in electron diffraction experiments, where dose is historically quoted in electrons/Å2 rather than in gray (Gy), and finally the development of a RADDOSE-3D GUI, enabling easy access to all the options available in the program.

Estimate your dose: 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.

RABDAM: quantifying specific radiation damage in individual protein crystal structures.

Radiation damage remains one of the major limitations to accurate structure determination in protein crystallography (PX). Despite the use of cryo-cooling techniques, it is highly probable that a number of the structures deposited in the Protein Data Bank (PDB) have suffered substantial radiation damage as a result of the high flux densities of third generation synchrotron X-ray sources. Whereas the effects of global damage upon diffraction pattern reflection intensities are readily detectable, traditionally the (earlier onset) site-specific structural changes induced by radiation damage have proven difficult to identify within individual PX structures. More recently, however, development of the BDamage metric has helped to address this problem. BDamage is a quantitative, per-atom metric identifies potential sites of specific damage by comparing the atomic B-factor values of atoms that occupy a similar local packing density environment in the structure. Building upon this past work, this article presents a program, RABDAM, to calculate the BDamage metric for all selected atoms within any standard-format PDB or mmCIF file. RABDAM provides several useful outputs to assess the extent of damage suffered by an input PX structure. This free and open-source software will allow assessment and improvement of the quality of PX structures both previously and newly deposited in the PDB.

Plant cysteine oxidases are dioxygenases that directly enable arginyl transferase-catalysed arginylation of N-end rule targets.

Crop yield loss due to flooding is a threat to food security. Submergence-induced hypoxia in plants results in stabilization of group VII ETHYLENE RESPONSE FACTORs (ERF-VIIs), which aid survival under these adverse conditions. ERF-VII stability is controlled by the N-end rule pathway, which proposes that ERF-VII N-terminal cysteine oxidation in normoxia enables arginylation followed by proteasomal degradation. The PLANT CYSTEINE OXIDASEs (PCOs) have been identified as catalysts of this oxidation. ERF-VII stabilization in hypoxia presumably arises from reduced PCO activity. We directly demonstrate that PCO dioxygenase activity produces Cys-sulfinic acid at the N terminus of an ERF-VII peptide, which then undergoes efficient arginylation by an arginyl transferase (ATE1). This provides molecular evidence of N-terminal Cys-sulfinic acid formation and arginylation by N-end rule pathway components, and a substrate of ATE1 in plants. The PCOs and ATE1 may be viable intervention targets to stabilize N-end rule substrates, including ERF-VIIs, to enhance submergence tolerance in agriculture.