CHESS Users ’ Meeting 2013 Tribute to Sol Gruner - CHESS Director 1996 - 2013 June 4 - 5 , 2013

Small angle X-ray scattering (SAXS) is an increasingly popular technique for obtaining low resolution solution structures of dynamic macromolecules and complexes. Because SAXS does not require special sample preparation beyond that needed to ensure monodispersity, it is commonly used to probe the dependence of macromolecular conformation or association on different solution conditions (such as temperature, pH, and ionic strength). Such combinatorial experiments are often limited by consumption of both sample and synchrotron time. Therefore, for high throughput applications of SAXS, it is desirable to find ways to economize sample and to accelerate data collection. Although modern X-ray sources can deliver sufficient flux for millisecond data acquisition in micron-scale samples, the high sensitivity to radiation damage of macromolecules in solution demands that the total dose be distributed over a large volume (typically >10 microliters), either by defocusing the beam or flowing the sample through it. Furthermore, frequent, aggressive cleaning of X-ray windows of the sample cell is often required due, for example to protein adsorption. Experience from electron microscopy (EM) and X-ray crystallography (MX) suggests that the maximum allowable radiation dose may be significantly larger at low temperatures than at ambient conditions. In addition to reducing radiation damage, cryocooling prevents evaporation of the sample droplet, and therefore “window-free” sample holders may be used. While cryocooling is now standard practice for EM and MX, it has not yet been embraced by the SAXS community. Because of the difficulty in producing homogeneous and reproducibly vitrified solutions, the requirement of accurately measuring and subtracting the solvent background scattering for SAXS was seen as incompatible with cryocooling. From a series of experiments performed at CHESS beamlines F2, C1, and G1, we identified cryoprotectants that produce homogeneously vitrified droplets by rapid cooling in a 100 K gas stream. Scattering profiles from similarly vitrified, macromolecule-containing solutions resemble those acquired at room temperature. The ability to expose for longer periods of time before damaging the sample more than compensates for the reduction in signal to background resulting from the cryoprotectants. As a result of increased dose tolerance, the sample volume can be reduced by orders of magnitude relative to room temperature. We collect cryo-SAXS data of sufficient quality to determine molecular shape reconstructions from illuminated volumes as small as 100 nanoliters. Studies to date show minimal artifacts introduced by