A Workflow for Protein Structure Determination from Thin Crystal Lamella by Micro-Electron Diffraction
A Workflow for Protein Structure Determination from Thin Crystal Lamella by Micro-Electron Diffraction
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通过微电子衍射从薄晶层测定蛋白质结构的工作流程
DOI：
10.1101/2020.04.30.061895
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Beale E
中科院分区：
文献类型：
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作者：
Beale E
近年来，微电子能谱已成为分析原子分辨率生物结构的一种强有力的方法。这项技术在很大程度上局限于蛋白质纳米晶体，这些纳米晶体要么长得像针，要么长成平板，厚度只有几百纳米。此外，传统的microED数据处理使用现有的X射线结晶学软件，该软件没有针对处理电子衍射数据特有的化合物效应进行优化。在这里，我们介绍了一套完整的microED工作流程，从低温聚焦离子束研磨制备样品，到使用标准CETA-D探测器进行数据采集，再到使用DIALS软件套件进行数据处理，从而实现了使用microED对任何大小和形状的蛋白质晶体进行常规原子结构测定。我们通过确定蛋白水解酶K的结构来证明该工作流程的有效性，并展示了使用蛋白质晶体片层相对于纳米晶体的优势。
MicroED has recently emerged as a powerful method for the analysis of biological structures at atomic resolution. This technique has been largely limited to protein nanocrystals which grow either as needles or plates measuring only a few hundred nanometers in thickness. Furthermore, traditional microED data processing uses established X-ray crystallography software that is not optimized for handling compound effects that are unique to electron diffraction data. Here, we present an integrated workflow for microED, from sample preparation by cryo-focused ion beam milling, through data collection with a standard Ceta-D detector, to data processing using the DIALS software suite, thus enabling routine atomic structure determination of protein crystals of any size and shape using microED. We demonstrate the effectiveness of the workflow by determining the structure of proteinase K to 2.0 Å resolution and show the advantage of using protein crystal lamellae over nanocrystals.