Abstract
Radiation damage represents a fundamental limit in the determination of protein structures via macromolecular crystallography (MX) at third-generation synchrotron sources. Over the past decade, improvements in both source and detector technology have led to MX experiments being performed with smaller and smaller crystals (on the order of a few microns), often using microfocus beams. Under these conditions, photoelectrons (PEs), the primary agents of radiation-damage in MX, may escape the diffraction volume prior to depositing all of their energy. The impact of PE escape is more significant at higher beam energies (>20 keV) as the electron inelastic mean free path (IMFP) is longer, allowing the electrons to deposit their energy over a larger area, extending further from their point of origin. Software such as RADDOSE-3D has been used extensively to predict the dose (energy absorbed per unit mass) that a crystal will absorb under a given set of experimental parameters and is an important component in planning a successful MX experiment. At the time this study was undertaken, dose predictions made using RADDOSE-3D were spatially-resolved, but did not yet account for the propagation of PEs through the diffraction volume. Hence, in the case of microfocus crystallography, it is anticipated that deviations may occur between the predicted and actual dose absorbed due to the influence of PEs. To explore this effect, we conducted a series of simulations of the dose absorbed by micron-sized crystals during microfocus MX experiments. Our simulations spanned beam and crystal sizes ranging from 1μm to 5μm for beam energies between 9 keV and 30 keV. Our simulations were spatially and temporarily resolved and accounted for the escape of PEs from the diffraction volume. The spatially-resolved dose maps produced by these simulations were used to predict the rate of intensity loss in a Bragg spot, a key metric for tracking global radiation damage. Our results were compared to predictions obtained using a recent version of RADDOSE-3D that did not account for PE escape; the predicted crystal lifetimes are shown to differ significantly for the smallest crystals and for high-energy beams, when PE escape is included in the simulations.
Highlights
Technological advances over the last few decades have helped to mitigate, though not eliminate, radiation damage in protein crystallography
We used 2D dose maps produced according to Section 4.5, and 3D dose maps produced using RADDOSE-3D [9], as inputs for a spot-fading model (Section 4.7) that simulated the loss in integrated intensity of a 3 Å Bragg spot under a range of beam- and sample-conditions
The results of our spot-fading model, comparing the effects of variation in crystal size, beam size, and beam energy; calculations of the change in X-ray interaction cross-sections under our simulation parameters, and the implications this has for diffraction efficiency in micro-crystallography; and a representation of a 2D slice through the real-space diffraction volume, illustrating how the contribution to diffracted intensity within this volume evolves as the crystal absorbs a dose of up to 60 MGy
Summary
Technological advances over the last few decades have helped to mitigate, though not eliminate, radiation damage in protein crystallography. They have facilitated the use of smaller, less perfect crystals in structure determination. Experiments can be performed using microfocus X-ray beams with sufficient flux density to cause rapid degradation of crystals. The use of such highly-focused beams is Crystals 2018, 8, 267; doi:10.3390/cryst8070267 www.mdpi.com/journal/crystals. The characteristics of small crystals, combined with these developments, create new challenges for understanding the effects of radiation damage. For an overview of the field, the reader is directed toward the recent reviews by Garman [3,4,5,6] and Holton [7]
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