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The collection of MicroED data for macromolecular crystallography

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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.

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Figure 1: Structures determined by MicroED.
Figure 2: Flow diagram for MicroED data collection protocol.
Figure 3: Representative protein microcrystal images.
Figure 4: Assembled Vitrobot coolant container.
Figure 5: Setting the eucentric height correctly.
Figure 6: Low-dose settings menu on the FEI Tecnai F20 microscope.
Figure 7: Example images from search, focus and exposure modes from a model MicroED sample.

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Acknowledgements

Work in the Gonen lab is supported by the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA

    Dan Shi, Brent L Nannenga, M Jason de la Cruz, Jinyang Liu, Steven Sawtelle, Francis E Reyes, Johan Hattne & Tamir Gonen

  2. Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

    Guillermo Calero

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  1. Dan Shi
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  2. Brent L Nannenga
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  3. M Jason de la Cruz
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  4. Jinyang Liu
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  5. Steven Sawtelle
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  6. Guillermo Calero
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  7. Francis E Reyes
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  8. Johan Hattne
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  9. Tamir Gonen
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Contributions

D.S., B.L.N. and T.G. wrote the manuscript. D.S., B.L.N., M.J.d.l.C., F.E.R., J.H. and T.G. designed and performed the experiments, and edited the manuscript. J.L. and J.H. worked on software design. D.S., T.G. and S.S. designed and built the rotation controller. G.C. provided Fig. 2d,h. All authors read and approved the manuscript.

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Correspondence to Tamir Gonen.

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The authors declare no competing financial interests.

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Shi, D., Nannenga, B., de la Cruz, M. et al. The collection of MicroED data for macromolecular crystallography. Nat Protoc 11, 895–904 (2016). https://doi.org/10.1038/nprot.2016.046

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