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Free keywords:
SOFT X-RAYS; DATA-COLLECTION; MACROMOLECULAR CRYSTALLOGRAPHY; ANOMALOUS
DIFFRACTION; ATOMIC-RESOLUTION; PROTEIN CRYSTALS; STRUCTURAL BASIS;
ROUTINE USE; OXIDASE; FAMILYChemistry; Crystallography; Materials Science; single-wavelength anomalous dispersion; native-SAD phasing; Se/S-SAD;
UV-laser cutting; crystal shaping; spherical crystals; absorption
correction; anomalous scattering factor; structure determination;
Abstract:
Native single-wavelength anomalous dispersion (SAD) is an attractive experimental phasing technique as it exploits weak anomalous signals from intrinsic light scatterers (Z < 20). The anomalous signal of sulfur in particular, is enhanced at long wavelengths, however the absorption of diffracted X-rays owing to the crystal, the sample support and air affects the recorded intensities. Thereby, the optimal measurable anomalous signals primarily depend on the counterplay of the absorption and the anomalous scattering factor at a given X-ray wavelength. Here, the benefit of using a wavelength of 2.7 over 1.9 angstrom is demonstrated for native-SAD phasing on a 266 kDa multiprotein-ligand tubulin complex (T2R-TTL) and is applied in the structure determination of an 86 kDa helicase Sen1 protein at beamline BL-1A of the KEK Photon Factory, Japan. Furthermore, X-ray absorption at long wavelengths was controlled by shaping a lysozyme crystal into spheres of defined thicknesses using a deep-UV laser, and a systematic comparison between wavelengths of 2.7 and 3.3 angstrom is reported for native SAD. The potential of laser-shaping technology and other challenges for an optimized native-SAD experiment at wavelengths >3 angstrom are discussed.
