Radiation damage and dose limits in serial synchrotron crystallography at cryo- and room temperatures

Abstract

Radiation damage limits the accuracy of macromolecular structures in X-ray crystallography. Cryogenic (cryo-) cooling reduces the global radiation damage rate and, therefore, became the method of choice over the past decades. The recent advent of serial crystallography, which spreads the absorbed energy over many crystals, thereby reducing damage, has rendered room temperature (RT) data collection more practical and also extendable to microcrystals, both enabling and requiring the study of specific and global radiation damage at RT. Here, we performed sequential serial raster-scanning crystallography using a microfocused synchrotron beam that allowed for the collection of two series of 40 and 90 full datasets at 2- and 1.9-Å resolution at a dose rate of 40.3 MGy/s on hen egg white lysozyme (HEWL) crystals at RT and cryotemperature, respectively. The diffraction intensity halved its initial value at average doses (D1/2) of 0.57 and 15.3 MGy at RT and 100 K, respectively. Specific radiation damage at RT was observed at disulfide bonds but not at acidic residues, increasing and then apparently reversing, a peculiar behavior that can be modeled by accounting for differential diffraction intensity decay due to the nonuniform illumination by the X-ray beam. Specific damage to disulfide bonds is evident early on at RT and proceeds at a fivefold higher rate than global damage. The decay modeling suggests it is advisable not to exceed a dose of 0.38 MGy per dataset in static and time-resolved synchrotron crystallography experiments at RT. This rough yardstick might change for proteins other than HEWL and at resolutions other than 2 Å.

Serial femtosecond and serial synchrotron crystallography yield data of equivalent quality: a systematic comparison

“…This implies that only in very critical cases radiation damage is a crucial criterion for the choice of the radiation source. While the present SSX data were collected at doses of approximately 170 kGy, with new rapid acquisition detectors (Eiger) it is possible to collect SSX data with < 2 ms exposure time thus at much lower doses (< 5kGy), further mitigating the differences between SFX and SSX data 18 .…”

“…However, while radiation damage is more pronounced at synchrotron sources, much of its deleterious effects can be mitigated by using low-dose exposure, which still leaves the electron density interpretable with little ambiguity 16,17 . The dose causing a 50% drop in overall diffraction intensity for lysozyme at room temperature SSX has been reported to be 380 kGy 18 . While this represents the upper dose limit for serial crystallography experiments, which should not be exceeded at room temperature, the onset of sitespecific damage could be observed at a much lower dose of approximately 80 kGy 18 .…”

“…The dose causing a 50% drop in overall diffraction intensity for lysozyme at room temperature SSX has been reported to be 380 kGy 18 . While this represents the upper dose limit for serial crystallography experiments, which should not be exceeded at room temperature, the onset of sitespecific damage could be observed at a much lower dose of approximately 80 kGy 18 . Although this value is much lower than doses encountered during typical cryo experiments, at modern microfocus beamlines it is still possible to collect multiple frames from a single micro-crystal at room temperature within this dose limit [17][18][19] .…”

SAXS studies of X-ray induced disulfide bond damage: Engineering high-resolution insight from a low-resolution technique

“…However, cryo-cooling can also limit biologically meaningful conformations [8,9] and produce structural artefacts [10,11]. For these reasons there has been interest in a return to near physiological temperature data collection and the corresponding development of serial crystallography methods to limit radiation damage [12].…”

“…The X-ray doses used for structural studies are on the order of tens of kilo-grays up to mega-grays and different damage processes have been observed in cryogenic and near physiological temperature crystallographic experiments [7,[12][13][14]. Damage mechanisms at cryogenic temperature have been well studied at large X-ray doses, but how relevant they are in physiologic conditions (i.e.…”