<em>In situ</em> protein micro-crystal fabrication by cryo-FIB for electron diffraction

Xinmei Li; Shuangbo Zhang; Jianguo Zhang; Fei Sun

doi:10.1007/s41048-018-0075-x

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Xinmei Li, Shuangbo Zhang, Jianguo Zhang, Fei Sun. In situ protein micro-crystal fabrication by cryo-FIB for electron diffraction. Biophysics Reports, 2018, 4(6): 339-347. doi: 10.1007/s41048-018-0075-x

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Xinmei Li, Shuangbo Zhang, Jianguo Zhang, Fei Sun. In situ protein micro-crystal fabrication by cryo-FIB for electron diffraction. Biophysics Reports, 2018, 4(6): 339-347. doi: 10.1007/s41048-018-0075-x

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

doi: 10.1007/s41048-018-0075-x

Xinmei Li^1,2,
Shuangbo Zhang^1,2,
Jianguo Zhang³,
Fei Sun^{1,2,3
,
,}

1 National Key Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
2 University of Chinese Academy of Sciences, Beijing 100049, China
3 Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China

Funds: This work was supported by grants from Chinese Academy of Sciences (ZDKYYQ20170002 and XDB08030202) and the Ministry of Science and Technology of China (2017YFA0504700 and 2014CB910700).

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Corresponding author: Fei Sun
Received Date: 27 July 2018
Rev Recd Date: 30 August 2018
Publish Date: 31 December 2018

Abstract

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 completeworkflowto prepare suitable crystals efficiently for MicroED experiment.Thisworkflow 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.
- Cryo-electron microscopy,
- Cryo focused ion beam,
- Electron diffraction,
- In situ crystallization,
- Microcrystal

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References(38)

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