Predicting the X-ray lifetime of protein crystals

Abstract

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 ...

Radiation decay of thaumatin crystals at three X-ray energies

“…In a recent study (Zeldin et al, 2013), D 50 values varied from 7 to 42 MGy depending on how the dose was estimated. The authors conclude that the 'diffraction-weighted dose' is the most efficient metric, and report a value of 12.9 MGy for insulin crystals at a resolution of 1.8 Å , which is also significantly lower than the value reported by Owen and coworkers.…”

Assessing and maximizing data quality in macromolecular crystallography

“…Strategic considerations regarding other aspects of data collection (e.g. [1,2]) and data reduction (e.g. [3,4••]) are also important, but are beyond the scope of this review.…”

RNA protects a nucleoprotein complex against radiation damage

“…Significant progress has been made in recent years in understanding the inevitable manifestations of X-ray-induced RD within protein crystals, and there is now a body of literature on possible strategies to mitigate the effects of RD (e.g. Zeldin, Brockhauser et al, 2013;Bourenkov & Popov, 2010). However, there is still no general consensus within the field on how to minimize RD during MX data collection, and debates on the dependence of RD progression on incident X-ray energy (Shimizu et al, 2007;Liebschner et ISSN 2059-7983 al., 2015) and the efficacy of radical scavengers (Allan et al, 2013) have yet to be resolved.…”

“…The beam-intensity profile was modelled as a uniform ('top-hat') distribution. The diffraction-weighted dose (DWD) values (Zeldin, Brockhauser et al, 2013) are given in Supplementary Table S1.…”