Time-resolved serial femtosecond crystallography (TR-SFX) at an x-ray free electron laser enables protein structural changes to be imaged on time-scales from femtoseconds to seconds. It can, however, be difficult to grasp the nature and timescale of global protein motions when structural changes are not isolated near a single active site. New tools are, therefore, needed to represent the global nature of electron density changes and their correlation with modeled protein structural changes. Here, we use TR-SFX data from bacteriorhodopsin to develop and validate a method for quantifying time-dependent electron density changes and correlating them throughout the protein. We define a spherical volume of difference electron density about selected atoms, average separately the positive and negative electron difference densities within each volume, and walk this spherical volume through all atoms within the protein. By correlating the resulting difference electron density amplitudes with time, our approach facilitates an initial assessment of the number and timescale of structural intermediates and highlights quake-like motions on the sub-picosecond timescale. This tool also allows structural models to be compared with experimental data using theoretical difference electron density changes calculated from refined resting and photo-activated structures.

中文翻译：

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在时间分辨晶体学中可视化蛋白质运动的工具

X射线自由电子激光的时间分辨串联飞秒晶体学（TR-SFX）使蛋白质结构变化可以在从飞秒到秒的时间范围内成像。但是，如果没有在单个活性位点附近隔离结构变化，可能很难把握全局蛋白质运动的性质和时间尺度。因此，需要新的工具来表示电子密度变化的整体性质及其与建模的蛋白质结构变化的相关性。在这里，我们使用来自细菌视紫红质的TR-SFX数据来开发和验证一种量化随时间变化的电子密度变化并将其与整个蛋白质相关联的方法。我们定义一个围绕所选原子的差分电子密度的球形体积，分别平均每个体积内的正负电子密度，并使该球形体积穿过蛋白质中的所有原子。通过将产生的差异电子密度幅度与时间相关联，我们的方法有助于初步评估结构中间体的数量和时标，并在亚皮秒级时标上突出显示类似地震的运动。该工具还允许使用从精确的静止和光激活结构计算出的理论差电子密度变化，将结构模型与实验数据进行比较。