Radiation damage in protein serial femtosecond crystallography using an x-ray free-electron laser

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Radiation damage is a major cause of failure in macromolecular crystallography experiments. Although it is always best to evenly illuminate the entire volume of a homogeneously diffracting crystal, limitations of the available equipment and imperfections in the sample often require a more sophisticated targeting strategy, involving microbeams smaller than the crystal, and translations of the crystal during data collection. This leads to a highly inhomogeneous distribution of absorbed X-rays (i.e., dose). Under these common experimental conditions, the relationship between dose and time is nonlinear, making it difficult to design an experimental strategy that optimizes the radiation damage lifetime of the crystal, or to assign appropriate dose values to an experiment. We present, and experimentally validate, a predictive metric diffraction-weighted dose for modeling the rate of decay of total diffracted intensity from protein crystals in macromolecular crystallography, and hence we can now assign appropriate "dose" values to modern experimental setups. Further, by taking the ratio of total elastic scattering to diffraction-weighted dose, we show that it is possible to directly compare potential data-collection strategies to optimize the diffraction for a given level of damage under specific experimental conditions. As an example of the applicability of this method, we demonstrate that by offsetting the rotation axis from the beam axis by 1.25 times the full-width half maximum of the beam, it is possible to significantly extend the dose lifetime of the crystal, leading to a higher number of diffracted photons, better statistics, and lower overall radiation damage.G iven an adequately diffracting crystal, radiation damage is the dominant cause of failure for macromolecular crystallography (MX) experiments (1), and overcoming this problem has been one of the major motivations for the development of new methods. By way of illustration: over the last 11 years at beamline 8.3.1 of the Advanced Light Source (ALS), more than 1,000 structures have been solved and deposited into the Protein Data Bank, but more than 25,000 datasets were collected. Similar dataset-to-deposition ratios have been reported elsewhere (2, 3). A retrospective analysis of the ALS 8.3.1 data reveals that radiation damage played a dominant role in the failure to obtain phases for structure solution by anomalous dispersion methods. Indeed, if radiation damage did not exist, investigators could simply keep collecting data until any desired signal-to-noise ratio was attained.Much of the recent excitement over serial femtosecond crystallography with X-ray Free Electron Lasers (XFELs) has been due to the vast gains in the diffraction/damage ratios demonstrated (4). Despite these major advances, the technology for these systems is not yet mature, and the linear nature of the XFEL facilities limits the number of end stations, greatly reducing capacity compared with a traditional synchrotron source. Synchrotron-based MX is thus likely to remain the dominant ...

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confidence: 99%

Predicting the X-ray lifetime of protein crystals

Zeldin

Brockhauser

Bremridge

et al. 2013

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“…Central to this method is the "diffraction before destruction" (1) process in which a still diffraction image is produced by a single X-ray pulse before significant radiation-induced electronic and atomic rearrangements occur within the crystal (1)(2)(3). At the Linac Coherent Light Source (LCLS) at SLAC, a single ∼50-fs-long X-ray pulse can expose a crystal to as many X-ray photons as a typical synchrotron beam line produces in about a second.…”

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confidence: 99%

Goniometer-based femtosecond crystallography with X-ray free electron lasers

Cohen

Soltis

González

et al. 2014

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128

The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiationsensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. For smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β 2 -adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.femtosecond diffraction | crystallography | XFEL | structural biology U sing extremely bright, short-timescale X-ray pulses produced by X-ray free-electron lasers (XFELs), femtosecond crystallography (FX) is an emerging method that expands the structural information accessible from very small or very radiation-sensitive macromolecular crystals. Central to this method is the "diffraction before destruction" (1) process in which a still diffraction image is produced by a single X-ray pulse before significant radiation-induced electronic and atomic rearrangements occur within the crystal (1-3). At the Linac Coherent Light Source (LCLS) at SLAC, a single ∼50-fs-long X-ray pulse can expose a crystal to as many X-ray photons as a typical synchrotron beam line produces in about a second. Exposing small crystals to these intense ultrashort pulses circumvents the dose limitations of conventional X-ray diffraction experiments (4) and may produce useful data to resolutions beyond what is achievable at synchrotrons (5). This innovation provides a pathway to obtain atomic information from proteins that only form micrometer-to nanometer-sized crystals, such as many membrane proteins and large multiprotein complexes. Moreover, XFELs enable "diffraction before reduction" data collection to address another major challenge in structural enzymology by providing a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzyme active sites (6), such as high-valency reaction intermediates that may be significantly photoreduced during a single X-ray exposure at a synchrotron, even at very small doses (7-11). Furthermore, the use of short (tens of femtoseconds) X-ray pulses further complements the structural characterization of biochemical reaction processes by providing access to a time domain two to three orders of magnitude faster (12, 13) than currently accessible using synchrotro...

“…Given the high dose per crystal used in the SFX measurements, we looked for evidence of predicted local radiation damage (13,14) in the vicinity of radiation-sensitive disulfide bonds. Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Simulations (20,21) and experiments (12)(13)(14) suggest that it should be possible to outrun radiation damage with short enough pulses using much higher X-ray intensities. Calculations with pulse durations as short as 10 fs predict the possibility of high-resolution structure determination from nanocrystals at doses greater than 10 GGy, and 1-TGy doses should be tolerable with subfemtosecond pulses (22).…”

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confidence: 99%

“…Serial femtosecond crystallography (SFX) overcomes radiation damage by using ultrabright X-ray pulses that are shorter in duration than most damage processes (9)(10)(11)(12)(13)(14), thereby vastly increasing the tolerable dose and thus the amount of information gathered before the specimen is destroyed. Commonly, crystals in a liquid stream are injected into the X-ray beam, and each crystal diffracts for only a few femtoseconds before being destroyed (12,15).…”

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confidence: 99%

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Atomic structure of granulin determined from native nanocrystalline granulovirus using an X-ray free-electron laser

Gati

Oberthüer

Yefanov

et al. 2017

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To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense femtosecond-duration pulses from X-ray free-electron lasers (XFELs) can outrun most damage processes, vastly increasing the tolerable dose before the specimen is destroyed. This in turn allows structure determination from crystals much smaller and more radiation sensitive than previously considered possible, allowing data collection from room temperature structures and avoiding structural changes due to cooling. Regardless, high-resolution structures obtained from XFEL data mostly use crystals far larger than 1 μm 3 in volume, whereas the X-ray beam is often attenuated to protect the detector from damage caused by intense Bragg spots. Here, we describe the 2 Å resolution structure of native nanocrystalline granulovirus occlusion bodies (OBs) that are less than 0.016 μm 3 in volume using the full power of the Linac Coherent Light Source (LCLS) and a dose up to 1.3 GGy per crystal. The crystalline shell of granulovirus OBs consists, on average, of about 9,000 unit cells, representing the smallest protein crystals to yield a high-resolution structure by X-ray crystallography to date. The XFEL structure shows little to no evidence of radiation damage and is more complete than a model determined using synchrotron data from recombinantly produced, much larger, cryocooled granulovirus granulin microcrystals. Our measurements suggest that it should be possible, under ideal experimental conditions, to obtain data from protein crystals with only 100 unit cells in volume using currently available XFELs and suggest that single-molecule imaging of individual biomolecules could almost be within reach.XFEL | nanocrystals | structural biology | bioimaging | SFX I maging of biomolecules using radiation of short enough wavelength to resolve individual atoms is limited by radiation damage, which destroys the very structure being measured. Energy absorption is unavoidable because the ratio of elastic scattering to damaging photoabsorption is an inherent property of atoms. Absorbed dose is therefore proportional to the scattered intensity, and thus the measured signal, used to determine the structure. The maximum tolerable dose that the sample can withstand fundamentally limits atomic resolution structure determination (1). The tolerable dose, and therefore damage, is a function of spatial resolution, with fine detail being destroyed first (2, 3). Radiation damage is typically overcome by distributing the dose over many identical copies of the same molecule, either using crystals containing many aligned copies of the same molecule, or by aligning images of individual molecules in the case of single particle electron microscopy. At room temperature, free radical production after a dose as small as 150 kGy (gray = J _ kg −1 absorbed energy) causes rapid decay of biological samples (4). This decay can be reduced by cooling the sample to below ∼120 K....