Instrumentation and experimental procedures for robust collection of X-ray diffraction data from protein crystals across physiological temperatures
Instrumentation and experimental procedures for robust collection of X-ray diffraction data from protein crystals across physiological temperatures
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DOI：
10.1107/s1600576720013503
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发表时间：
2020-12-01
影响因子：
6.1
通讯作者：
Yabukarski, Filip
中科院分区：
文献类型：
--
作者：
Doukov, Tzanko;Herschlag, Daniel;Yabukarski, Filip
在低温下收集的传统X射线衍射数据为蛋白质的三维结构提供了宝贵的见解，为结构-功能研究提供了支柱。虽然低温可以减轻辐射损伤，但低温可以改变蛋白质的构象和溶剂结构。此外，构象系综是蛋白质功能和能量学的基础，室温X射线结晶学的最新进展提供了构象异质性信息，这些信息可以直接与生物功能相关。鉴于这种能力，下一个挑战是开发一种可靠和广泛适用的方法来收集室温及以上的单晶X射线衍射数据。这一挑战在这里得到了解决。所描述的方法提供了完整的衍射数据集，总收集时间短至从单个蛋白质晶体中收集5s，大大增加了在分配的同步加速器束时间内可以收集的数据量。通过在斯坦福同步辐射光源的BL14-1上收集293-363K温度范围内的1.09-1.54埃分辨率数据，证明了它的适用性。本文的分析表明，衍射数据质量高，不受过度脱水或辐射损伤的影响。
Traditional X-ray diffraction data collected at cryo-temperatures have delivered invaluable insights into the three-dimensional structures of proteins, providing the backbone of structure-function studies. While cryo-cooling mitigates radiation damage, cryo-temperatures can alter protein conformational ensembles and solvent structure. Furthermore, conformational ensembles underlie protein function and energetics, and recent advances in room-temperature X-ray crystallography have delivered conformational heterogeneity information that can be directly related to biological function. Given this capability, the next challenge is to develop a robust and broadly applicable method to collect single-crystal X-ray diffraction data at and above room temperature. This challenge is addressed herein. The approach described provides complete diffraction data sets with total collection times as short as similar to 5s from single protein crystals, dramatically increasing the quantity of data that can be collected within allocated synchrotron beam time. Its applicability was demonstrated by collecting 1.09-1.54 angstrom resolution data over a temperature range of 293-363K for proteinase K, thaumatin and lysozyme crystals at BL14-1 at the Stanford Synchrotron Radiation Lightsource. The analyses presented here indicate that the diffraction data are of high quality and do not suffer from excessive dehydration or radiation damage.