The collection of MicroED data for macromolecular crystallography

Dan Shi, Brent L. Nannenga, M. Jason De La Cruz, Jinyang Liu, Steven Sawtelle, Guillermo Calero, Francis E. Reyes, Johan Hattne, Tamir Gonen

Research output: Contribution to journalArticle

51 Scopus citations

Abstract

The formation of large, well-ordered crystals for crystallographic experiments remains a crucial bottleneck to the structural understanding of many important biological systems. To help alleviate this problem in crystallography, we have developed the MicroED method for the collection of electron diffraction data from 3D microcrystals and nanocrystals of radiation-sensitive biological material. In this approach, liquid solutions containing protein microcrystals are deposited on carbon-coated electron microscopy grids and are vitrified by plunging them into liquid ethane. MicroED data are collected for each selected crystal using cryo-electron microscopy, in which the crystal is diffracted using very few electrons as the stage is continuously rotated. This protocol gives advice on how to identify microcrystals by light microscopy or by negative-stain electron microscopy in samples obtained from standard protein crystallization experiments. The protocol also includes information about custom-designed equipment for controlling crystal rotation and software for recording experimental parameters in diffraction image metadata. Identifying microcrystals, preparing samples and setting up the microscope for diffraction data collection take approximately half an hour for each step. Screening microcrystals for quality diffraction takes roughly an hour, and the collection of a single data set is ∼10 min in duration. Complete data sets and resulting high-resolution structures can be obtained from a single crystal or by merging data from multiple crystals.

Original languageEnglish (US)
Pages (from-to)895-904
Number of pages10
JournalNature protocols
Volume11
Issue number5
DOIs
StatePublished - May 1 2016

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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  • Cite this

    Shi, D., Nannenga, B. L., De La Cruz, M. J., Liu, J., Sawtelle, S., Calero, G., Reyes, F. E., Hattne, J., & Gonen, T. (2016). The collection of MicroED data for macromolecular crystallography. Nature protocols, 11(5), 895-904. https://doi.org/10.1038/nprot.2016.046