Data collection with a tailored X-ray beam size at 2.69 angstrom wavelength (4.6 keV): sulfur SAD phasing of Cdc23(Nterm)

摘要

The capability to reach wavelengths of up to 3.1 angstrom 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 lambda = 2.69 angstrom (4.6 keV) allowed the crystal structure determination by sulfur SAD phasing of Cdc23(Nterm), a subunit of the multimeric anaphase-promoting complex (APC/C). At this energy, Cdc23(Nterm) has an expected Bijvoet ratio <|F-anom|>/< 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 mu m in diameter allowed crystal twinning to be overcome. The crystals diffracted to 3.1 angstrom resolution, with unit-cell parameters a = b = 61.2, c = 151.5 angstrom, and belonged to space group P4(3). The refined structure to 3.1 angstrom resolution has an R factor of 18.7% and an R-free of 25.9%. This paper reports the structure solution, related methods and a discussion of the instrumentation.