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Method | Open Access

In situ protein micro-crystal fabrication by cryo-FIB for electron diffraction

Xinmei Li1,2Shuangbo Zhang1,2Jianguo Zhang3Fei Sun1,2,3( )
National Key Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
University of Chinese Academy of Sciences, Beijing 100049, China
Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
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Abstract

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 in situ 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 μm3. Our work will greatly expand the availability space of crystals suitable for MicroED and fill up the gap between MicroED and X-ray crystallography.

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Biophysics Reports
Pages 339-347
Cite this article:
Li X, Zhang S, Zhang J, et al. In situ protein micro-crystal fabrication by cryo-FIB for electron diffraction. Biophysics Reports, 2018, 4(6): 339-347. https://doi.org/10.1007/s41048-018-0075-x

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Received: 27 July 2018
Accepted: 30 August 2018
Published: 14 November 2018
© The Author(s) 2018

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

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