Accessing protein conformational ensembles using room-temperature X-ray crystallography
Accessing protein conformational ensembles using room-temperature X-ray crystallography
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DOI：
10.1073/pnas.1111325108
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发表时间：
2011-09-27
影响因子：
11.1
通讯作者：
Alber, Tom
中科院分区：
文献类型：
--
作者：
Fraser, James S.;van den Bedem, Henry;Alber, Tom
现代蛋白质晶体结构几乎完全基于在低温（通常为100 K）下收集的X射线数据。冷却过程被认为在结构结果的功能解释中引入很少的偏差，因为低温对整个蛋白质骨架折叠的干扰最小。相比之下，在这里，我们表明，闪冷偏见以前隐藏在蛋白质晶体的结构合奏。通过使用新的计算工具进行电子密度采样，模型改进和分子堆积分析，分析了30种不同蛋白质的可用数据，我们发现晶体低温冷却重塑了超过35%的侧链的构象分布，并消除了功能运动所需的堆积缺陷。在信号开关蛋白H-Ras中，通过NMR在溶液中检测到的波动一致的变构网络在室温下被发现，但在低温下，电子密度图中没有。这些结果暴露了结构数据库偏向于更小，过度包装和不切实际的独特模型。通过X射线晶体学监测室温构象系综可以揭示对催化、配体结合和变构调节至关重要的运动。
Modern protein crystal structures are based nearly exclusively on X-ray data collected at cryogenic temperatures (generally 100 K). The cooling process is thought to introduce little bias in the functional interpretation of structural results, because cryogenic temperatures minimally perturb the overall protein backbone fold. In contrast, here we show that flash cooling biases previously hidden structural ensembles in protein crystals. By analyzing available data for 30 different proteins using new computational tools for electron-density sampling, model refinement, and molecular packing analysis, we found that crystal cryocooling remodels the conformational distributions of more than 35% of side chains and eliminates packing defects necessary for functional motions. In the signaling switch protein, H-Ras, an allosteric network consistent with fluctuations detected in solution by NMR was uncovered in the room-temperature, but not the cryogenic, electron-density maps. These results expose a bias in structural databases toward smaller, overpacked, and unrealistically unique models. Monitoring room-temperature conformational ensembles by X-ray crystallography can reveal motions crucial for catalysis, ligand binding, and allosteric regulation.