Oxidation states of individual metal atoms within a metalloprotein can be assigned by examining X-ray absorption edges, which shift to higher energy for progressively more positive valence numbers. Indeed, X-ray crystallography is well suited for such a measurement, owing to its ability to spatially resolve the scattering contributions of individual metal atoms that have distinct electronic environments contributing to protein function. However, as the magnitude of the shift is quite small, about +2 eV per valence state for iron, it has only been possible to measure the effect when performed with monochromated X-ray sources at synchrotron facilities with energy resolutions in the range 2-3 × 10-4 (ΔE/E). This paper tests whether X-ray free-electron laser (XFEL) pulses, which have a broader bandpass (ΔE/E = 3 × 10-3) when used without a monochromator, might also be useful for such studies. The program nanoBragg is used to simulate serial femtosecond crystallography (SFX) diffraction images with sufficient granularity to model the XFEL spectrum, the crystal mosaicity and the wavelength-dependent anomalous scattering factors contributed by two differently charged iron centers in the 110-amino-acid protein, ferredoxin. Bayesian methods are then used to deduce, from the simulated data, the most likely X-ray absorption curves for each metal atom in the protein, which agree well with the curves chosen for the simulation. The data analysis relies critically on the ability to measure the incident spectrum for each pulse, and also on the nanoBragg simulator to predict the size, shape and intensity profile of Bragg spots based on an underlying physical model that includes the absorption curves, which are then modified to produce the best agreement with the simulated data. This inference methodology potentially enables the use of SFX diffraction for the study of metalloenzyme mechanisms and, in general, offers a more detailed approach to Bragg spot data reduction.

中文翻译：

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通过 XFEL 晶体衍射的详细建模来实现金属蛋白电荷态的空间分辨率。


金属蛋白内单个金属原子的氧化态可以通过检查 X 射线吸收边来确定，X 射线吸收边会转向更高的能量以获得逐渐更多的正价数。事实上，X 射线晶体学非常适合这种测量，因为它能够在空间上解析单个金属原子的散射贡献，这些金属原子具有对蛋白质功能有不同的电子环境。然而，由于偏移的幅度非常小，对于铁来说每价态大约 +2 eV，因此只有在同步加速器设施中使用单色 X 射线源进行测量时才能测量效果，能量分辨率范围为 2- 3×10-4（ΔE/E）。本文测试了 X 射线自由电子激光 (XFEL) 脉冲是否也可用于此类研究，该脉冲在不使用单色仪的情况下具有更宽的带通 (ΔE/E = 3 × 10-3)。 NanoBragg 程序用于模拟连续飞秒晶体学 (SFX) 衍射图像，具有足够的粒度来模拟 XFEL 光谱、晶体镶嵌性以及由 110 个氨基酸蛋白质中两个带不同电荷的铁中心贡献的波长依赖性异常散射因子，铁氧还蛋白。然后使用贝叶斯方法从模拟数据中推导出蛋白质中每个金属原子最可能的 X 射线吸收曲线，该曲线与为模拟选择的曲线非常吻合。数据分析主要依赖于测量每个脉冲的入射光谱的能力，以及纳米布拉格模拟器根据包括吸收曲线在内的基础物理模型来预测布拉格斑点的大小、形状和强度分布，然后将其进行修改以产生与模拟数据的最佳一致性。 这种推理方法有可能使 SFX 衍射用于金属酶机制的研究，并且通常为布拉格点数据简化提供了更详细的方法。