Abstract
High-quality high-multiplicity X-ray diffraction data were collected on five different crystals of thaumatin using a homogeneous-profile X-ray beam at E = 8 keV to investigate the counteracting effects of increased multiplicity and increased radiation damage on the quality of anomalous diffraction data collected on macromolecular crystals. By comparing sulfur substructures obtained from subsets of the data selected as a function of absorbed X-ray dose with sulfur positions in the respective refined reference structures, the doses at which the highest quality of anomalous differences could be obtained were identified for the five crystals. A statistic σ{ΔF}D, calculated as the width σ of the normalized distribution of a set {ΔF} of anomalous differences collected at a dose D, is suggested as a measure of anomalous data quality as a function of dose. An empirical rule is proposed to identify the dose at which the gains in data quality due to increased multiplicity are outbalanced by the losses due to decreases in signal-to-noise as a consequence of radiation damage. Identifying this point of diminishing returns allows the optimization of the choice of data collection parameters and the selection of data to be used in subsequent crystal structure determination steps.
Highlights
Single-anomalous dispersion phasing (SAD) exploiting the anomalous signal from sulfur atoms (S-SAD) has become a widely applied method in macromolecular crystallography (Hendrickson, 2014; Liu & Hendrickson, 2015)
We address the problem of identifying the point of diminishing returns in high-multiplicity S-SAD phasing, i.e. the point at which the inclusion of additional data from a progressively more damaged crystal leads to a deterioration in the quality of the substructure determined from these data, from only experimental diffraction data in a systematic fashion by correlating statistical properties of the measured anomalous differences with the quality of the substructure obtained
Based on the above observations, we propose that the effect of radiation damage on the quality of anomalous difference can be measured by evaluating {ÁF} for independent groups of reflections as the deposited X-ray dose increases
Summary
Single-anomalous dispersion phasing (SAD) exploiting the anomalous signal from sulfur atoms (S-SAD) has become a widely applied method in macromolecular crystallography (Hendrickson, 2014; Liu & Hendrickson, 2015). An important advantage of S-SAD phasing is that the S atoms providing the anomalous signal are naturally present in many protein molecules obviating the need to introduce anomalous scatterers such as selenium or metal atoms into the crystal. The expected anomalous signal is generally in the few percent range and can be difficult to measure. It has been recognized early on that the accuracy of the measurements of small anomalous differences, and the chances of solving the corresponding structure by S-SAD phasing, can be improved by collecting data with high multiplicity (Dauter & Adamiak, 2001; Weiss et al, 2001). Even with the most accurate experimental apparatus, the collection of high-multiplicity diffraction data from a macromolecular crystal is limited by X-ray radiation damage (Garman & Weik, 2017)
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