Radiation damage is a major cause of failure in macromolecular crystallography experiments. Although it is always best to evenly illuminate the entire volume of a homogeneously diffracting crystal, limitations of the available equipment and imperfections in the sample often require a more sophisticated targeting strategy, involving microbeams smaller than the crystal, and translations of the crystal during data collection. This leads to a highly inhomogeneous distribution of absorbed X-rays (i.e., dose). Under these common experimental conditions, the relationship between dose and time is nonlinear, making it difficult to design an experimental strategy that optimizes the radiation damage lifetime of the crystal, or to assign appropriate dose values to an experiment. We present, and experimentally validate, a predictive metric diffraction-weighted dose for modeling the rate of decay of total diffracted intensity from protein crystals in macromolecular crystallography, and hence we can now assign appropriate "dose" values to modern experimental setups. Further, by taking the ratio of total elastic scattering to diffraction-weighted dose, we show that it is possible to directly compare potential data-collection strategies to optimize the diffraction for a given level of damage under specific experimental conditions. As an example of the applicability of this method, we demonstrate that by offsetting the rotation axis from the beam axis by 1.25 times the full-width half maximum of the beam, it is possible to significantly extend the dose lifetime of the crystal, leading to a higher number of diffracted photons, better statistics, and lower overall radiation damage.G iven an adequately diffracting crystal, radiation damage is the dominant cause of failure for macromolecular crystallography (MX) experiments (1), and overcoming this problem has been one of the major motivations for the development of new methods. By way of illustration: over the last 11 years at beamline 8.3.1 of the Advanced Light Source (ALS), more than 1,000 structures have been solved and deposited into the Protein Data Bank, but more than 25,000 datasets were collected. Similar dataset-to-deposition ratios have been reported elsewhere (2, 3). A retrospective analysis of the ALS 8.3.1 data reveals that radiation damage played a dominant role in the failure to obtain phases for structure solution by anomalous dispersion methods. Indeed, if radiation damage did not exist, investigators could simply keep collecting data until any desired signal-to-noise ratio was attained.Much of the recent excitement over serial femtosecond crystallography with X-ray Free Electron Lasers (XFELs) has been due to the vast gains in the diffraction/damage ratios demonstrated (4). Despite these major advances, the technology for these systems is not yet mature, and the linear nature of the XFEL facilities limits the number of end stations, greatly reducing capacity compared with a traditional synchrotron source. Synchrotron-based MX is thus likely to remain the dominant ...
A common challenge for macromolecular crystallographers is to solve a difficult crystal structure from one well-diffracting crystal amongst many poorly diffracting ones. This presentation is about how to make the best possible use of this limited diffraction volume using a newly developed metric: Diffraction Weighted Dose.Radiation damage during the diffraction experiment fundamentally limits how much data can be collected from a given crystal, and can degrade the image quality or even entirely prevent an atomic model from being obtained (1).Extensive work has been carried out to understand the dose-dependent decay of diffraction quality (2-5), and guidelines exist for upper limits on the acceptable dose under carefully controlled even dose conditions. However, when applied to 'real world' scenarios, where the strategy has to be optimised for data collection rather than for systematic investigations of radiation damage progression, these results are not generally applicable (6). For a typical data collection, with non-uniform illumination from an approximately Gaussian X-ray beam and crystal rotation, the dose is not distributed evenly throughout the crystal volume. Thus the guidelines developed for a single dose state resulting from a uniform beam profile are difficult to apply.We present (i) results from using the program RADDOSE-3D (7) to optimally distribute dose within a crystal volume, and (ii) a new metric: Diffraction Weighted Dose, which faithfully describes the relative total diffraction efficiency (the loss of intensity relative to the original diffraction intensity of the crystal: I n /I 1 ) for a dataset collected from an un-evenly exposed crystal. Together, these developments represent a vital step towards eliminating radiation damage as a source of experimental failure for macromolecular crystallography.
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