Li Xinmei, Zhang Shuangbo, Zhang Jianguo, Sun Fei
1National Key Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China.
2University of Chinese Academy of Sciences, Beijing, 100049 China.
Biophys Rep. 2018;4(6):339-347. doi: 10.1007/s41048-018-0075-x. Epub 2018 Nov 14.
Micro-electron diffraction (MicroED) is an emerging technique to use cryo-electron microscope to study the crystal structures of macromolecule from its micro-/nano-crystals, which are not suitable for conventional X-ray crystallography. However, this technique has been prevented for its wide application by the limited availability of producing good micro-/nano-crystals and the inappropriate transfer of crystals. Here, we developed a complete workflow to prepare suitable crystals efficiently for MicroED experiment. This workflow includes on-grid crystallization, single-side blotting, cryo-focus ion beam (cryo-FIB) fabrication, and cryo-electron diffraction of crystal cryo-lamella. This workflow enables us to apply MicroED to study many small macromolecular crystals with the size of 2-10 μm, which is too large for MicroED but quite small for conventional X-ray crystallography. We have applied this method to solve 2.5 Å crystal structure of lysozyme from its micro-crystal within the size of 10 × 10 × 10 μm. Our work will greatly expand the availability space of crystals suitable for MicroED and fill up the gap between MicroED and X-ray crystallography.
微电子衍射（MicroED）是一种利用低温电子显微镜从微/纳米晶体研究大分子晶体结构的新兴技术，这些微/纳米晶体不适用于传统的X射线晶体学。然而，由于难以获得高质量的微/纳米晶体以及晶体转移不当，该技术的广泛应用受到了限制。在此，我们开发了一套完整的工作流程，能够高效地制备适合MicroED实验的晶体。该工作流程包括在网格上结晶、单面印迹、低温聚焦离子束（cryo-FIB）制备以及晶体低温薄片的低温电子衍射。这一工作流程使我们能够应用MicroED研究许多尺寸为2-10μm的小型大分子晶体，这些晶体对于MicroED来说太大，但对于传统X射线晶体学来说又太小。我们已应用此方法解析了尺寸为10×10×10μm的溶菌酶微晶的2.5Å晶体结构。我们的工作将极大地拓展适合MicroED的晶体的可得空间，并填补MicroED与X射线晶体学之间的空白。
