The Influence of Photoelectron Escape in Radiation Damage Simulations of Protein Micro-Crystallography

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 1 µm to 5 µm 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.

Doses for experiments with microbeams and microcrystals: Monte Carlo simulations in RADDOSE‐3D

“…The Nave and Hill simulations also suggested that the effect of photoelectron escape is more significant at higher incident X‐ray energies ( E inc ), and a similar method was employed by Cowan and Nave 14 to suggest a significant advantage in collecting data at E inc higher than the ~12.4 keV routinely used (20–30 keV E inc was predicted to be optimum) for small crystals, providing other experimental factors remain optimized (e.g., detector efficiency etc.). More recent Monte Carlo simulations 15–17 have arrived at similar conclusions, and the simulations from Dickerson et al . predicted a significant improvement on increasing incident energy from 12.4 to 26 keV for crystals of 5 μm or smaller, provided that a detector efficient at these higher energies is used 17 .…”

“…The Nave and Hill simulations also suggested that the effect of photoelectron escape is more significant at higher incident X-ray energies (E inc ), and a similar method was employed by Cowan and Nave 14 to suggest a significant advantage in collecting data at E inc higher than the 12.4 keV routinely used (20-30 keV E inc was predicted to be optimum) for small crystals, providing other experimental factors remain optimized (e.g., detector efficiency etc.). More recent Monte Carlo simulations [15][16][17] have arrived at similar conclusions, and the simulations from Dickerson et al predicted a significant improvement on increasing incident energy from 12.4 to 26 keV for crystals of 5 μm or smaller, provided that a detector efficient at these higher energies is used. 17 Experimentally, Sanishvili et al 18 demonstrated reduced radiation damage rates in F I G U R E 1 Range of electrons travelling through amorphous ice, calculated using the CSDA protein crystals using micron-sized 15.1 keV and 18.5 keV X-ray beams and reconciled these results with Monte Carlo simulations to demonstrate the extent of the escape of photoelectrons from the diffracting volume of the crystal.…”

Megahertz pulse trains enable multi-hit serial femtosecond crystallography experiments at X-ray free electron lasers

“…The RADDOSE-3D version 4 (X-FEL) 39 gave an estimated dose of 0.2 MGy, taking into account the photo electron escape and the FW of the beam. The second method gave an estimate of 0.165 MGy maximum dose per pulse received by the crystal within the beam using the approach of Marman et al 51 . which includes photo electron escape and in addition specifically the absorbed dose as a function of the crystal position within the incident beam (Supplementary Fig.…”

“…The first method is based on Monte-Carlo modelling of the primary photoelectron trajectories, taking in account any photoelectron escape from the crystal that might occur subsequent to the crystal interacting with the X-ray pulse. This approach to determining the dose absorbed by the crystal was adapted from a discrete simulation of radiation damage model based on Marman et al 51 . The X-ray beam was modelled as a symmetric 2-dimensional Lorentzian distribution with a full width of 65.5 µm (accounting for 99% of the X-ray flux).…”

X-ray radiation damage to biological samples: recent progress

“…These calculations take into account the energy response of the CdTe detector as well as the probability of photoelectron escape from the irradiated crystal volume and entry of photoelectrons from any irradiated surrounding material. As has previously been suggested, photoelectron escape can potentially reduce the absorbed dose (Nave & Hill, 2005;Cowan & Nave, 2008;Marman et al, 2018) and thus improve the DE. The results presented here identify a maximum in the DE at a 'sweet spot' of 26 keV if using crystals of 5 mm or less with a microbeam which is either matched in size to (or smaller than) the crystal.…”