Statistically correcting dynamical electron scattering improves the refinement of protein nanocrystals, including charge refinement of coordinated metals.
Statistically correcting dynamical electron scattering improves the refinement of protein nanocrystals, including charge refinement of coordinated metals.
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DOI：
10.1107/s2059798320014540
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发表时间：
2021-01-01
期刊：
影响因子：
--
通讯作者：
Abrahams JP
中科院分区：
文献类型：
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作者：
Blum TB;Housset D;Clabbers MTB;van Genderen E;Bacia-Verloop M;Zander U;McCarthy AA;Schoehn G;Ling WL;Abrahams JP
改进了基于可能性的方法来校正弱衍射蛋白质纳米晶体中的动态散射。该方法已在三种蛋白质晶体上进行了测试，并产生了更好的细化统计数据和结构解释，包括结合离子的电荷估计。当只有纳米尺寸的晶体可用时，电子衍射可以确定蛋白质结构。然而，在电子衍射中很突出的多重弹性（或动态）散射是一个令人担忧的问题。目前通过多层或布洛赫波方法模拟动态散射的方法不适合蛋白质晶体，因为它们不是为处理大分子而设计的。在这里，通过从薄晶体收集数据来限制胰岛素、嗜热菌蛋白酶和索马甜纳米晶体的动态散射。为了准确测量来自薄晶体中少数晶胞的微弱衍射信号，使用了低噪声混合像素 Timepix 电子计数探测器。使用基于可能性的校正进一步减少了剩余的动力学分量，该校正之前是为了分析小分子纳米晶体的电子衍射数据而引入的，并且在这里适用于蛋白质晶体。该过程被证明可以显着改善结构细化，在一种情况下允许溶剂分子的定位。它还可以通过金属原子及其配体的 B 因子分析来细化结合金属原子（蛋白质功能中的重要元素）的电荷状态。这些结果显然增加了大分子电子晶体学作为补充结构生物学技术的价值。
A likelihood-based method is improved to correct for dynamical scattering in weakly diffracting protein nanocrystals. The method has been tested on three protein crystals and yielded better refinement statistics and structure interpretations, including charge estimations of bound ions. Electron diffraction allows protein structure determination when only nanosized crystals are available. Nevertheless, multiple elastic (or dynamical) scattering, which is prominent in electron diffraction, is a concern. Current methods for modeling dynamical scattering by multi-slice or Bloch wave approaches are not suitable for protein crystals because they are not designed to cope with large molecules. Here, dynamical scattering of nanocrystals of insulin, thermolysin and thaumatin was limited by collecting data from thin crystals. To accurately measure the weak diffraction signal from the few unit cells in the thin crystals, a low-noise hybrid pixel Timepix electron-counting detector was used. The remaining dynamical component was further reduced in refinement using a likelihood-based correction, which was introduced previously for analyzing electron diffraction data of small-molecule nanocrystals and was adapted here for protein crystals. The procedure is shown to notably improve the structural refinement, in one case allowing the location of solvent molecules. It also allowed refinement of the charge states of bound metal atoms, an important element in protein function, through B-factor analysis of the metal atoms and their ligands. These results clearly increase the value of macromolecular electron crystallography as a complementary structural biology technique.