Mitigation of X-ray damage in macromolecular crystallography by submicrometre line focusing

Finfrock, Y. Zou; Stern, Edward A.; Alkire, R. W.; Kas, J. J.; Evans‐Lutterodt, K.; Stein, Aaron; Duke, N.E.C.; Lazarski, K.; Joachimiak, A.

doi:10.1107/s0907444913009335

Cited by 15 publications

(18 citation statements)

References 34 publications

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“…The previous observation by Sanishvili et al indicated the presence of perturbations extending a few micrometers beyond the X-ray-exposed regions (12), consistent with the proposed redistribution of charge and the production of strong local electric fields (14,15). The experimentally measured parameters describing the mean propagation distance of the photoelectrons based on the SHG images are in remarkably good quantitative agreement with the Monte Carlo simulations.…”

Section: Resultssupporting

confidence: 76%

Imaging local electric fields produced upon synchrotron X-ray exposure

Dettmar

Newman

Toth

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Electron-hole separation following hard X-ray absorption during diffraction analysis of soft materials under cryogenic conditions produces substantial local electric fields visualizable by second harmonic generation (SHG) microscopy. Monte Carlo simulations of X-ray photoelectron trajectories suggest the formation of substantial local electric fields in the regions adjacent to those exposed to X-rays, indicating a possible electric-field-induced SHG (EFISH) mechanism for generating the observed signal. In studies of amorphous vitreous solvents, analysis of the SHG spatial profiles following X-ray microbeam exposure was consistent with an EFISH mechanism. Within protein crystals, exposure to 12-keV (1.033-Å) X-rays resulted in increased SHG in the region extending ∼3 μm beyond the borders of the X-ray beam. Moderate X-ray exposures typical of those used for crystal centering by raster scanning through an X-ray beam were sufficient to produce static electric fields easily detectable by SHG. The X-ray-induced SHG activity was observed with no measurable loss for longer than 2 wk while maintained under cryogenic conditions, but disappeared if annealed to room temperature for a few seconds. These results provide direct experimental observables capable of validating simulations of X-ray-induced damage within soft materials. In addition, X-ray-induced local fields may potentially impact diffraction resolution through localized piezoelectric distortions of the lattice.synchrotron | EFISH | X-ray damage | piezoelectric | structural biology T he ability to determine atomic-resolution structures and the quality of resulting structures recovered by X-ray diffraction can be profoundly affected by X-ray induced damage during the diffraction data acquisition. At the energies typically used for protein structure determination, for every X-ray photon resulting in elastic scattering approximately 10-fold more photons result in inelastic scattering or absorption and deposition of energy into the crystal. The excess energy can perturb the molecules within a crystal through a variety of different mechanisms, typically resulting in the reduction of overall diffracted intensity (1). With biological macromolecules, such as proteins, this effect in combination with the initial inherent degree of ordering often limits the achievable resolution possible through signal averaging.X-ray induced radiation damage in protein crystals can manifest as two different types: global and specific (1). Specific damage occurs when irradiation causes specific chemical changes within the protein such as loss of side chains or disulfide bridge linkages (2, 3), damage to active sites (2), and damage to metal centers within the protein (1). Disulfide anion radicals, evidence of specific damage, have previously been observed by UV-vis microspectrophotometry after X-ray exposure for tens of milliseconds (4). Damage to nonspecific sites, known also as global damage, results in a decrease in the intensity of the high-angle spots with increasing X-ray dose, and t...

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Section: Resultssupporting

confidence: 76%

Imaging local electric fields produced upon synchrotron X-ray exposure

Dettmar

Newman

Toth

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Our findings suggest that in some cases the lack of inclusion of PE escape leads to deviations from the behaviour predicted by RADDOSE-3D [9] (see, for example, [14,16,17]). This conclusion is supported by both the similarity in output to RADDOSE-3D under conditions where PE escape is unlikely to affect dose (or when it has been ignored in our models), and the divergence in output under conditions where PE escape is expected to be significant.…”

Section: Prediction Of Extended Lifetimes For Small Crystals and Beammentioning

confidence: 77%

“…The similarity in values of f NH indicate that our simulation results are comparable to the Holton and Frankel model. In addition to qualitative comparisons to previous modelling studies, a semi-quantitative assessment of our model can be conducted by comparison to recent results obtained by Finfrock et al [16,18] and Sanishvili et al [17], who performed experimental studies of radiation damage in large crystals by exposing sub-volumes using micro-focus and line-focus beams. They observed that the radiation damage footprint extended between 1.5 µm and 5 µm beyond the actual beam footprint, showing the spread of PEs beyond the diffraction volume.…”

Section: Simulations Enabling Qualitative and Semi-quantitative Intermentioning

confidence: 99%

“…Several experiments [16][17][18] have shown reduced damage occurring due to PE escape, but have only explored a narrow parameter space thus far; a more thorough examination, particularly focussed on the benefit of using higher energy beams, could be guided by our results and data obtained could subsequently be used to validate and improve our model. Should experiments show quantitative agreement with our predictions, our model could be used to provide more accurate predictions of dose rate, and therefore crystal lifetime in micro-crystallography experiments, compared to currently available radiation damage software packages.…”

Section: Micro-crystallography Could Benefit Significantly From Highementioning

confidence: 99%

“…Suggestions generally rely on making the beam footprint smaller (in at least one dimension) than the damage footprint. This may be accomplished by using microfocus or line-focus X-ray beams [16][17][18]. Some special cases have also been investigated, where PE escape might be enhanced, these include imaging of platelet or needle-like crystals [22].…”

Section: Introductionmentioning

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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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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Mitigation of X-ray damage in macromolecular crystallography by submicrometre line focusing

Cited by 15 publications

References 34 publications

Imaging local electric fields produced upon synchrotron X-ray exposure

Imaging local electric fields produced upon synchrotron X-ray exposure

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

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

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