Long-wavelength native-SAD phasing: opportunities and challenges
Long-wavelength native-SAD phasing: opportunities and challenges
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DOI：
10.1107/s2052252519002756
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发表时间：
2019-05-01
期刊：
影响因子：
3.9
通讯作者：
Wang, Meitian
中科院分区：
文献类型：
--
作者：
Basu, Shibom;Olieric, Vincent;Wang, Meitian
自然单波长反常色散（SAD）是一种有吸引力的实验定相技术，因为它利用来自本征光散射体（Z < 20）的弱反常信号。特别是硫的异常信号在长波长处增强，然而由于晶体、样品支撑物和空气对衍射X射线的吸收影响记录的强度。因此，最佳的可测量的异常信号主要取决于在给定的X射线波长处的吸收和异常散射因子的相互作用。在这里，使用2.7埃的波长超过1.9埃的好处被证明为266 kDa的多蛋白质-配体微管蛋白复合物（T2 R-TTL）上的天然SAD定相，并在86 kDa的解旋酶Sen 1蛋白在光束线BL-1A的KEK光子工厂，日本的结构测定中应用。此外，在长波长的X-射线吸收控制成形的溶菌酶晶体成球的定义的厚度使用深紫外激光，和系统的比较波长为2.7和3.3埃的报告为本地SAD。讨论了激光整形技术的潜力和波长>3埃的优化本机SAD实验的其他挑战。
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.