Measurements of accurate x-ray scattering data of protein solutions using small stationary sample cells

Abstract

In this paper, we report a method of precise in situ x-ray scattering measurements on protein solutions using small stationary sample cells. Although reduction in the radiation damage induced by intense synchrotron radiation sources is indispensable for the correct interpretation of scattering data, there is still a lack of effective methods to overcome radiation-induced aggregation and extract scattering profiles free from chemical or structural damage. It is found that radiation-induced aggregation mainly begins on the surface of the sample cell and grows along the beam path; the diameter of the damaged region is comparable to the x-ray beam size. Radiation-induced aggregation can be effectively avoided by using a two-dimensional scan ͑2D mode͒, with an interval as small as 1.5 times the beam size, at low temperature ͑e.g., 4°C͒. A radiation sensitive protein, bovine hemoglobin, was used to test the method. A standard deviation of less than 5% in the small angle region was observed from a series of nine spectra recorded in 2D mode, in contrast to the intensity variation seen using the conventional stationary technique, which can exceed 100%. Wide-angle x-ray scattering data were collected at a standard macromolecular diffraction station using the same data collection protocol and showed a good signal/noise ratio ͑better than the reported data on the same protein using a flow cell͒. The results indicate that this method is an effective approach for obtaining precise measurements of protein solution scattering.

Breaking the Radiation Damage Limit with Cryo-SAXS

“…Biophysical Journal 104(1) 227-236 SAXS from cryocooled glucose isomerase is insensitive to large x-ray doses X-ray-induced changes to the macromolecule's structure or solution state must be minimized to obtain reliable SAXS profiles. In room temperature solution SAXS, a series of profiles are acquired and inspected for dose and time-dependent changes using the radius of gyration as a means of quantifying damage and determining the maximum tolerable x-ray exposure (9,10). Collecting data on vitrified samples at T~100 K should eliminate radiation-induced aggregation (which otherwise dominates low-angle scattering) and reduce unfolding and fragmentation.…”

“…Biologically important targets such as metalloproteins and sensors can also exhibit fast damage at specific sites, e.g., at the enzymatically important metal site, that perturb ligand interactions and associated conformation changes. To minimize radiation doses and achieve adequate signal to noise, large sample volumes must be irradiated either by defocusing the x-ray beam (1) or by flowing (2)(3)(4) or translating (10) the sample through the beam. For a typical protein at 1 mg/mL concentration, the minimum sample consumption is roughly 12 mL (1).…”

Uridine as a new scavenger for synchrotron-based structural biology techniques

“…The cryo-SAXS method cryocools small volumes of sample, from $ 20 nL to $ 1 mL, to 100 K, allowing the proteins and nucleic acids to withstand doses up to 100-300 kGy. Other strategies for both techniques include attenuating or defocusing the X-ray beam (Hura et al, 2009;Schulze-Briese et al, 2005), flowing the sample continuously (Barty et al, 2012;Pernot et al, 2013;Martel et al, 2012;Nielsen et al, 2012) or translating the sample through the beam (Hong & Hao, 2009;Flot et al, 2010). Other approaches in SAXS include cooling the sample down to 4-10 C, which can be achieved with temperaturecontrolled cells coupled to automated collection systems (Hura et al, 2009;Pernot et al, 2013;Round et al, 2015;Blanchet et al, 2015), or reducing the exposure times (Fischetti et al, 2003;Pernot et al, 2013).…”

A microfabricated fixed path length silicon sample holder improves background subtraction for cryoSAXS

“…Owing to the small sample volume and the open top of the silicon sample holders, the sample evaporates too rapidly at room temperature to obtain reliable scattering profiles. Instead, acrylic sample holders similar to those described by Hong & Hao (2009) and with nominal sample volumes of $3 ml were laser cut and windowed with 25 mm-thick polystyrene (Goodfellow Corporation, Coraopolis, PA, USA). These sample holders screwed onto the same mounting apparatus used in the 100 K experiments.…”