Improving data quality and expanding BioSAXS experiments to low-molecular-weight and low-concentration protein samples.

Abstract

The addition of compounds to scavenge the radical species produced during biological small-angle X-ray scattering (BioSAXS) experiments is a common strategy to reduce the effects of radiation damage and produce better quality data. As almost half of the experiments leading to structures deposited in the SASBDB database used scavengers, finding potent scavengers would be advantageous for many experiments. Here, four compounds, three nucleosides and one nitrogenous base, are presented which can act as very effective radical-scavenging additives and increase the critical dose by up to 20 times without altering the stability or reducing the contrast of the tested protein solutions. The efficacy of these scavengers is higher than those commonly used in the field to date, as verified for lysozyme solutions at various concentrations from 7.0 to 0.5 mg ml-1. The compounds are also very efficient at mitigating radiation damage to four proteins with molecular weights ranging from 7 to 240 kDa and pH values from 3 to 8, with the extreme case being catalase at 6.7 mg ml-1, with a scavenging factor exceeding 100. These scavengers can therefore be instrumental in expanding BioSAXS to low-molecular-weight and low-concentration protein samples that were previously inaccessible owing to poor data quality. It is also demonstrated that an increase in the critical dose in standard BioSAXS experiments leads to an increment in the retrieved information, in particular at higher angles, and thus to higher resolution of the model.