Use of Cr Kα radiation to enhance the signal from anomalous scatterers including sulfur

The anomalous signals from scatterers such as sulfur (S) and arsenic (As) were compared in diffraction data sets collected from an X-ray source with three different targets, Au, Cu and Cr, on a multi-target rotating anode. HIV-1 integrase crystals served as the test case for this study. The crystalline specimen of HIV-1 integrase contains in each protein molecule two As atoms, each covalently bound to a cysteine S atom, and two additional S atoms derived from methionine. It was found that the Cr Kα radiation gave the clearest peaks in anomalous difference Fourier maps, although the signal-to-noise ratios of the anomalous signal for the Cu Kα and Cr Kα data were similar but better than that for Au Lα. This result was in spite 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 Cr Kα data were both peaks of the expected ellipsoidal shape, enveloping the As atom and the adjacent S atom. None of the S sites was apparent in difference maps calculated using the Au Lα data. The ability to enhance the S-derived anomalous signal using Cr Kα radiation has particularly useful applications in the structure determination of proteins, for example in resolving ambiguities in the chain tracing of a protein with numerous disulfide bonds and in assigning amino acid identities. Additionally, anomalous difference Patterson maps calculated from the Cr Kα data were sufficiently clear to identify the As-related peaks. These results form the groundwork for in-house phase determination with the multi-wavelength anomalous diffraction method.