De novo crystal structure determination of double stranded RNA binding domain using only the sulfur anomalous diffraction in SAD phasing

Beatriz Gomes Guimarães; Béatrice Golinelli-Pimpaneau

doi:10.1016/j.crstbi.2021.05.002

De novo crystal structure determination of double stranded RNA binding domain using only the sulfur anomalous diffraction in SAD phasing

Curr Res Struct Biol. 2021 Jun 6:3:112-120. doi: 10.1016/j.crstbi.2021.05.002. eCollection 2021.

Authors

Beatriz Gomes Guimarães¹, Béatrice Golinelli-Pimpaneau²

Affiliations

¹ Synchrotron Soleil, L'Orme des Merisiers, Saint-Aubin 91190 Gif-sur-Yvette, France.
² Laboratoire de Chimie des Processus Biologiques, UMR 8229 CNRS, Collège de France, Sorbonne Université, 11 Place Marcelin Berthelot, 75005 Paris, France.

Abstract

Single-wavelength anomalous dispersion (SAD)-phasing using sulfur as the unique anomalous scatterer is a powerful method to solve the phase problem in protein crystallography. However, it is not yet widely used by non-expert crystallographers. We report here the structure determination of the double stranded RNA binding domain of human dihydrouridine synthase using the sulfur-SAD method and highly redundant data collected at 1.8 Å ("off-edge"), at which the estimated overall anomalous signal was 1.08%. High multiplicity data were collected on a single crystal rotated along the ϕ or ω axis at different κ angles, with the primary beam intensity being attenuated from 50% to 95%, compared to data collection at 0.98 Å, to reduce radiation damage. SHELXD succeeded to locate 14 out 15 sulfur sites only using the data sets recorded with highest beam attenuation, which provided phases sufficient for structure solving. In an attempt to stimulate the use of sulfur-SAD phasing by a broader community of crystallographers, we describe our experimental strategy together with a compilation of previous successful cases, suggesting that sulfur-SAD phasing should be attempted for determining the de novo structure of any protein with average sulfur content diffracting better than 3 Å resolution.

Keywords: Double stranded RNA binding Domain; Multi-orientation; S-SAD; Sulfur SAD-Phasing; de novo structure determination.