Use of CrK alpha radiation to enhance the signal from anomalous scatterersincluding sulfur

The anomalous signals from scatterers such as sulfur (S) and arsenic (As) w ere compared in diffraction data sets collected from an X-ray source with t hree different targets, Au, Cu and Cr, on a multi-target rotating anode. HI V-1 integrase crystals served as the test case for this study. The crystall ine specimen of HIV-1 integrase contains in each protein molecule two As at oms, each covalently bound to a cysteine S atom, and two additional S atoms derived from methionine. It was found that the Cr K alpha radiation gave t he clearest peaks in anomalous difference Fourier maps, although the signal -to-noise ratios of the anomalous signal for the Cu K alpha and Cr K alpha data were similar but better than that for Au L alpha. This result was in s pite of the fourfold higher flux from the Cu anode versus the Cr anode. For all three X-ray wavelengths, anomalous difference Fourier maps calculated with bias-removed phases derived from the known atomic model revealed clear peaks at the two As sites. However, only in the map calculated using the C r K alpha data were both peaks of the expected ellipsoidal shape, envelopin g the As atom and the adjacent S atom. None of the S sites was apparent in difference maps calculated using the Au L alpha data. The ability to enhanc e the S-derived anomalous signal using Cr K alpha radiation has particularl y useful applications in the structure determination of proteins, for examp le in resolving ambiguities in the chain tracing of a protein with numerous disulfide bonds and in assigning amino acid identities. Additionally, anom alous difference Patterson maps calculated from the Cr K alpha data were su fficiently clear to identify the As-related peaks. These results form the g roundwork for in-house phase determination with the multi-wavelength anomal ous diffraction method.