Instrumentation and experimental procedures for robust collection of X-ray diffraction data from protein crystals across physiological temperatures,Journal of Applied Crystallography

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Instrumentation and experimental procedures for robust collection of X-ray diffraction data from protein crystals across physiological temperatures
Journal of Applied Crystallography ( IF 5.2 ) Pub Date : 2020-11-05 , DOI: 10.1107/s1600576720013503
Tzanko Doukov ₁ , Daniel Herschlag _{2,

3,

4} , Filip Yabukarski ₂

Affiliation

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 ∼5 s 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 Å resolution data over a temperature range of 293-363 K 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.

中文翻译：

用于在生理温度下从蛋白质晶体中可靠收集 X 射线衍射数据的仪器和实验程序

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基础。虽然低温冷却可以减轻辐射损伤，但低温可以改变蛋白质构象整体和溶剂结构。此外，构象系综是蛋白质功能和能量学的基础，室温 X 射线晶体学的最新进展提供了与生物功能直接相关的构象异质性信息。鉴于这种能力，下一个挑战是开发一种稳健且广泛适用的方法来收集室温及室温以上的单晶 X 射线衍射数据。本文解决了这一挑战。所描述的方法提供了完整的衍射数据集，从单个蛋白质晶体的总收集时间短至〜5秒，显着增加了在分配的同步加速器光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 293-363 K 温度范围内的蛋白酶 K、索马甜和溶菌酶晶体的 1.09-1.54 Å 分辨率数据，证明了其适用性。这里提出的分析表明衍射数据是高质量的并且没有遭受过度脱水或辐射损伤。

更新日期：2020-11-05

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