Cryo-cooling has been nearly universally adopted to mitigate X-ray damage and facilitate crystal handling in protein X-ray crystallography. However, cryo X-ray crystallographic data provide an incomplete window into the ensemble of conformations that is at the heart of protein function and energetics. Room-temperature (RT) X-ray crystallography provides accurate ensemble information, and recent developments allow conformational heterogeneity (the experimental manifestation of ensembles) to be extracted from single-crystal data. Nevertheless, high sensitivity to X-ray damage at RT raises concerns about data reliability. To systematically address this critical issue, increasingly X-ray-damaged high-resolution data sets (1.02–1.52 Å resolution) were obtained from single proteinase K, thaumatin and lysozyme crystals at RT (277 K). In each case a modest increase in conformational heterogeneity with X-ray damage was observed. Merging data with different extents of damage (as is typically carried out) had negligible effects on conformational heterogeneity until the overall diffraction intensity decayed to ∼70% of its initial value. These effects were compared with X-ray damage effects in cryo-cooled crystals by carrying out an analogous analysis of increasingly damaged proteinase K cryo data sets (0.9–1.16 Å resolution). X-ray damage-associated heterogeneity changes were found that were not observed at RT. This property renders it difficult to distinguish real from artefactual conformations and to determine the conformational response to changes in temperature. The ability to acquire reliable heterogeneity information from single crystals at RT, together with recent advances in RT data collection at accessible synchrotron beamlines, provides a strong motivation for the widespread adoption of RT X-ray crystallography to obtain conformational ensemble information.

中文翻译：

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评估 X 射线损伤对室温 (277 K) 和低温冷却蛋白质晶体构象异质性的影响

冷冻冷却几乎已被普遍采用来减轻 X 射线损伤并促进蛋白质 X 射线晶体学中的晶体处理。然而，冷冻 X 射线晶体学数据为了解作为蛋白质功能和能量学核心的构象整体提供了一个不完整的窗口。室温 (RT) X 射线晶体学提供了准确的系综信息，最近的发展允许从单晶数据中提取构象异质性（系综的实验表现）。然而，RT 对 X 射线损伤的高敏感性引起了对数据可靠性的担忧。为了系统地解决这一关键问题，我们在室温 (277 K) 下从单一蛋白酶 K、索马甜和溶菌酶晶体中获得了越来越多的 X 射线损伤高分辨率数据集（1.02–1.52 Å 分辨率）。在每种情况下，都观察到 X 射线损伤导致构象异质性适度增加。合并具有不同损伤程度的数据（通常进行）对构象异质性的影响可以忽略不计，直到总体衍射强度衰减到其初始值的~70%。通过对日益受损的蛋白酶 K 冷冻数据集（0.9–1.16 Å 分辨率）进行类似分析，将这些效应与冷冻晶体中的 X 射线损伤效应进行比较。发现了 X 射线损伤相关的异质性变化，而在 RT 中未观察到。这一特性使得难以区分真实构象和人造构象，并难以确定对温度变化的构象响应。在 RT 下从单晶获取可靠的异质性信息的能力，加上在可访问的同步加速器光束线处 RT 数据收集的最新进展，为广泛采用 RT X 射线晶体学来获取构象系综信息提供了强大的动力。