Breaking the Radiation Damage Limit with Cryo-SAXS

Abstract

Small angle x-ray scattering (SAXS) is a versatile and widely used technique for obtaining low-resolution structures of macromolecules and complexes. SAXS experiments measure molecules in solution, without the need for labeling or crystallization. However, radiation damage currently limits the application of SAXS to molecules that can be produced in microgram quantities; for typical proteins, 10-20 μL of solution at 1 mg/mL is required to accumulate adequate signal before irreversible x-ray damage is observed. Here, we show that cryocooled proteins and nucleic acids can withstand doses at least two orders of magnitude larger than room temperature samples. We demonstrate accurate T = 100 K particle envelope reconstructions from sample volumes as small as 15 nL, a factor of 1000 smaller than in current practice. Cryo-SAXS will thus enable structure determination of difficult-to-express proteins and biologically important, highly radiation-sensitive proteins including light-activated switches and metalloenzymes.

Solid-liquid interfaces of ionic liquid solutions—Interfacial layering and bulk correlations

“…A typical x-ray dose soft matter samples might withstand is on the order of 400 Gy (lysozyme in aqueous solution) to 500 kGy (lysozyme crystal). 31 Therefore, we conclude that the stability of our IL sample with respect to x-ray radiation damage lies in the typical range for a soft matter system.…”

Combined small angle X-ray solution scattering with atomic force microscopy for characterizing radiation damage on biological macromolecules

“…However, it was also found that early effect of radiation damage was an increase of molecular size without any significant unfolding suggesting that radiation damage observed on BSA was compatible with the presence of radical activities [61]. Reduction in radiation damage has been obtained using Cryo-SAXS [12] or using time-resolved SAXS [62]. Besides, fast detection readout allows collection of SAXS before radiation damage occur [60].…”

“…When planning combined experiments, it is important to consider the effects of X-ray exposure, both short and long term, related to the bioSAXS experiment [11, 12]. BioSAXS data is routinely checked for short term variations during exposure and shows there is no variation on the length scales (low resolution size and shape) and time scales (1–10 s at dedicated facilities).…”

“…For example, radiation-induced aggregation has been observed with SAXS data for lysozyme, but without any change in folding topology [10]. Irreversible X-ray induced damage, essentially due to free radical formation in the sample at synchrotron sources, are a current limitation of SAXS experiments and often increase the amount of material needed [11] or require radiation protectant such as glycerol or cryo-cooling [12]. However, evaluation of post-SAXS experiment radiation damage on proteins is rarely performed because the allowable doses are highly sample-dependent, and must be determined on a case-by-case basis.…”

Emerging applications of small angle solution scattering in structural biology

“…Although SAS is essentially a low‐information technique, combining SAS with complementary techniques can circumvent this limitation in many cases . However, much work remains in method developments for data acquisition, analysis and model validation . In the coming years, modern X‐ray sources and methodological advances will probably open up unprecedented ways to reconstruct solution structures of biomacromolecules, and will conceivably reveal unforeseen wealth of information on protein motion, which will be a giant step forward …”