Data collection with a tailored X-ray beam size at 2.69 Å wavelength (4.6 keV): sulfur SAD phasing of Cdc23Nterm

Abstract

The capability to reach wavelengths of up to 3.1 Å at the newly established EMBL P13 beamline at PETRA III, the new third-generation synchrotron at DESY in Hamburg, provides the opportunity to explore very long wavelengths to harness the sulfur anomalous signal for phase determination. Data collection at λ = 2.69 Å (4.6 keV) allowed the crystal structure determination by sulfur SAD phasing of Cdc23Nterm, a subunit of the multimeric anaphase-promoting complex (APC/C). At this energy, Cdc23Ntermhas an expected Bijvoet ratio 〈|Fanom|〉/〈F〉 of 2.2%, with 282 residues, including six cysteines and five methionine residues, and two molecules in the asymmetric unit (65.4 kDa; 12 Cys and ten Met residues). Selectively illuminating two separate portions of the same crystal with an X-ray beam of 50 µm in diameter allowed crystal twinning to be overcome. The crystals diffracted to 3.1 Å resolution, with unit-cell parametersa=b= 61.2,c = 151.5 Å, and belonged to space groupP43. The refined structure to 3.1 Å resolution has anRfactor of 18.7% and anRfreeof 25.9%. This paper reports the structure solution, related methods and a discussion of the instrumentation.