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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DOI：
10.3389/fmolb.2020.00179
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发表时间：
2020-08-04
影响因子：
5
通讯作者：
Zhang, Peijun
中科院分区：
文献类型：
--
作者：
Beale, Emma, V;Waterman, David G.;Zhang, Peijun
MicroED最近已成为一种强大的方法，用于原子分辨率的生物结构分析。这项技术在很大程度上局限于蛋白质纳米晶体，这些纳米晶体生长为针或板，厚度仅为几百纳米。此外，传统的microED数据处理使用已建立的X射线晶体学软件，该软件未针对处理电子衍射数据所特有的化合物效应进行优化。在这里，我们提出了一个集成的工作流程microED，从样品制备冷冻聚焦离子束铣削，通过数据收集与标准Ceta-D检测器，数据处理使用DIALS软件套件，从而使常规原子结构测定蛋白质晶体的任何大小和形状使用microED。我们证明了工作流程的有效性，通过确定蛋白酶K的结构，以2.0埃的分辨率，并显示使用蛋白质晶体lamenium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 angstrom resolution and show the advantage of using protein crystal lamellae over nanocrystals.