Experimental phase determination with selenomethionine or mercury-derivatization in serial femtosecond crystallography.
Experimental phase determination with selenomethionine or mercury-derivatization in serial femtosecond crystallography.
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DOI：
10.1107/s2052252517008557
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发表时间：
2017-09-01
期刊：
影响因子：
3.9
通讯作者：
Nakatsu T
中科院分区：
文献类型：
--
作者：
Yamashita K;Kuwabara N;Nakane T;Murai T;Mizohata E;Sugahara M;Pan D;Masuda T;Suzuki M;Sato T;Kodan A;Yamaguchi T;Nango E;Tanaka T;Tono K;Joti Y;Kameshima T;Hatsui T;Yabashi M;Manya H;Endo T;Kato R;Senda T;Kato H;Iwata S;Ago H;Yamamoto M;Yumoto F;Nakatsu T
高能X射线是必不可少的从头结构测定与硒或汞的强烈异常散射。单波长异常衍射相位硒蛋氨酸衍生和汞浸泡技术已成功地应用于串行飞秒晶体学与13.0 keV或12.6 keV的X射线在SACLA生产。 使用X射线自由电子激光（XFEL）的连续飞秒晶体学（SFX）在蛋白质结构测定方面具有巨大的潜力，因为难以产生大的高质量晶体。SFX已被应用于各种系统，但很少用于具有先前未知结构的蛋白质。因此，大多数先前获得的SFX结构已经解决了分子置换法。为了便于蛋白质结构测定SFX，它是必不可少的，以建立定相方法，有效地为SFX。在这里，硒代甲硫氨酸衍生化和汞浸泡已被调查SFX实验使用高能量XFEL在SPring-8埃紧凑型自由电子激光器（SACLA），日本兵库县。报道了三个成功的单波长反常衍射（SAD）定相的案例，使用小于1 λ波长的X射线与合理的衍射图案数（13 000，60 000和11 000）。 它表明，从XFEL和常用的重原子掺入技术的高能量X射线的组合将使常规从头生物大分子的结构测定。
High-energy X-rays are essential for de novo structure determination with strong anomalous scattering from selenium or mercury. Single-wavelength anomalous diffraction phasing using selenomethionine-derivatization and mercury-soaking techniques has been successfully applied to serial femtosecond crystallography with 13.0 keV or 12.6 keV X-rays produced at SACLA. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) holds enormous potential for the structure determination of proteins for which it is difficult to produce large and high-quality crystals. SFX has been applied to various systems, but rarely to proteins that have previously unknown structures. Consequently, the majority of previously obtained SFX structures have been solved by the molecular replacement method. To facilitate protein structure determination by SFX, it is essential to establish phasing methods that work efficiently for SFX. Here, selenomethionine derivatization and mercury soaking have been investigated for SFX experiments using the high-energy XFEL at the SPring-8 Angstrom Compact Free-Electron Laser (SACLA), Hyogo, Japan. Three successful cases are reported of single-wavelength anomalous diffraction (SAD) phasing using X-rays of less than 1 Å wavelength with reasonable numbers of diffraction patterns (13 000, 60 000 and 11 000). It is demonstrated that the combination of high-energy X-rays from an XFEL and commonly used heavy-atom incorporation techniques will enable routine de novo structural determination of biomacromolecules.