Radiation Damage in Macromolecular Crystallography

Garman, Elspeth F.; Weik, Martin H.

doi:10.1080/08940886.2015.1101322

Cited by 14 publications

(37 citation statements)

References 139 publications

(142 reference statements)

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“…Previous studies [17,20,21] of the effect of PE escape have often incorporated Monte Carlo (MC) modelling of electron trajectories, performed using programmes such as CASINO [23,24]. In the current paper, we present the first systematic simulation study of PE escape in MX accounting for: (1) the effects of a finite beam size comparable to the PE stopping range; (2) the effects of a finite crystal size matched to the FWHM of the beam; (3) the effect of variation in X-ray beam energy; and (4) the influence of the beam profile as a function of the dose received by the crystal over time.…”

Section: Introductionmentioning

confidence: 99%

“…Cross-sections for coherent scatter, σ C , incoherent scatter, σ I , and photoelectric absorption, σ P , were calculated using the NIST XCOM database [25]. Diffraction efficiency was calculated as the ratio σ C /(σ I + σ P ) from cross-section data and the ratio of coherently scattered photons per unit volume, N C /σ 3 Figure 5 shows a series of 2D slices through the diffraction volume of a 1 µm crystal centred in a 12 keV, 1 µm X-ray beam for global doses ranging from 0 MGy to 60 MGy. We observe that at lower doses the shape of the diffraction volume is dominated by the Gaussian profile of the beam.…”

Section: Variation Of Diffraction Efficiency With Energymentioning

confidence: 99%

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

2018

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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.

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Section: Introductionmentioning

confidence: 99%

Section: Variation Of Diffraction Efficiency With Energymentioning

confidence: 99%

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

2018

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“…Cryocrystallography combined with the data-collection method of choice, for example, multi-or single-wavelength anomalous dispersion/diffraction (MAD or SAD respectively), is still the most common practice when measuring diffraction data. In fact, 90% of the structures deposited in the Protein Data Bank (as at 25 September 2017) were determined below 160 K [37]. However, macromolecules have been shown to lose some of their conformational states and functionality with cooling, distorting results, for example, in catalysis, ligand binding, allosteric regulation and time-resolved studies [38,39].…”

Section: Cryocrystallographymentioning

confidence: 99%

“…A top-hat profile beam is ideal in order to illuminate the crystal uniformly with the same flux across the irradiated volume and will result in a similar rate of damage throughout the crystal [37]. However, most commonly the beam shape has an approximately Gaussian profile, resulting in inhomogenous irradiation of the crystal.…”

Section: Data Collection Parametersmentioning

confidence: 99%

“…Therefore, the resulting electron density maps will be significantly noisier. Several global damage indicators can be plotted against absorbed dose to study radiation damage effects, for example, the decay of the average summed intensity of the data set over the initial average summed intensity (I n /I 1 ), the unit cell volume, and the relative scaling B-factor [29,37]. The processed data are then phased using the chosen method.…”

Section: Structure Solution and Interpretationmentioning

confidence: 99%

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Radiation Damage in Macromolecular Crystallography—An Experimentalist’s View

Taberman

2018

Crystals

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Radiation damage still remains a major limitation and challenge in macromolecular X-ray crystallography. Some of the high-intensity radiation used for diffraction data collection experiments is absorbed by the crystals, generating free radicals. These give rise to radiation damage even at cryotemperatures (~100 K), which can lead to incorrect biological conclusions being drawn from the resulting structure, or even prevent structure solution entirely. Investigation of mitigation strategies and the effects caused by radiation damage has been extensive over the past fifteen years. Here, recent understanding of the physical and chemical phenomena of radiation damage is described, along with the global effects inflicted on the collected data and the specific effects observed in the solved structure. Furthermore, this review aims to summarise the progress made in radiation damage studies in macromolecular crystallography from the experimentalist's point of view and to give an introduction to the current literature.

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

Dickerson

Garman

2020

Protein Science

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Increasingly, microbeams and microcrystals are being used for macromolecular crystallography (MX) experiments at synchrotrons. However, radiation damage remains a major concern since it is a fundamental limiting factor affecting the success of macromolecular structure determination. The rate of radiation damage at cryotemperatures is known to be proportional to the absorbed dose, so to optimize experimental outcomes, accurate dose calculations are required which take into account the physics of the interactions of the crystal constituents. The program RADDOSE‐3D estimates the dose absorbed by samples during MX data collection at synchrotron sources, allowing direct comparison of radiation damage between experiments carried out with different samples and beam parameters. This has aided the study of MX radiation damage and enabled prediction of approximately when it will manifest in diffraction patterns so it can potentially be avoided. However, the probability of photoelectron escape from the sample and entry from the surrounding material has not previously been included in RADDOSE‐3D, leading to potentially inaccurate does estimates for experiments using microbeams or microcrystals. We present an extension to RADDOSE‐3D which performs Monte Carlo simulations of a rotating crystal during MX data collection, taking into account the redistribution of photoelectrons produced both in the sample and the material surrounding the crystal. As well as providing more accurate dose estimates, the Monte Carlo simulations highlight the importance of the size and composition of the surrounding material on the dose and thus the rate of radiation damage to the sample. Minimizing irradiation of the surrounding material or removing it almost completely will be key to extending the lifetime of microcrystals and enhancing the potential benefits of using higher incident X‐ray energies.

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Radiation Damage in Macromolecular Crystallography

Cited by 14 publications

References 139 publications

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

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

Radiation Damage in Macromolecular Crystallography—An Experimentalist’s View

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

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