Native SAD Phasing at Room Temperature

A bstract Single-wavelength anomalous diffraction (SAD) is a routine method for overcoming the phase problem when solving a new macromolecular structure. This technique requires the accurate measurement of intensities to sensitively determine differences across Bijvoet pairs, making it a stringent test for the reliability of a data collection method. Although SAD experiments are commonly conducted at cryogenic temperatures to mitigate the effects of radiation damage, such temperatures can alter the conformational ensemble of the protein crystal and may impede the merging of data from multiple crystals due to non-uniform freezing. Here, we propose a data collection strategy to obtain high-quality data from room temperature samples. To illustrate the strengths of this approach we use native SAD phasing at 6.5 keV to solve four structures of three model systems at 295 K. The resulting datasets allow for automatic phasing and model building, and exhibit alternate conformations that are well-supported by the electron density. The high-redundancy data collection method demonstrated here enables the routine collection of high-quality, room-temperature diffraction to improve the study of protein conformational ensembles.