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Collection, pre-processing and on-the-fly analysis of data for high-resolution, single-particle cryo-electron microscopy

Abstract

The dramatic growth in the use of cryo-electron microscopy (cryo-EM) to generate high-resolution structures of macromolecular complexes has changed the landscape of structural biology. The majority of structures deposited in the Electron Microscopy Data Bank (EMDB) at higher than 4-Å resolution were collected on Titan Krios microscopes. Although the pipeline for single-particle data collection is becoming routine, there is much variation in how sessions are set up. Furthermore, when collection is under way, there are a range of approaches for efficiently moving and pre-processing these data. Here, we present a standard operating procedure for single-particle data collection with Thermo Fisher Scientific EPU software, using the two most common direct electron detectors (the Thermo Fisher Scientific Falcon 3 (F3EC) and the Gatan K2), as well as a strategy for structuring these data to enable efficient pre-processing and on-the-fly monitoring of data collection. This protocol takes 3–6 h to set up a typical automated data collection session.

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Fig. 1: Flowchart of the procedures.
Fig. 2: EPU setup.
Fig. 3: On-the-fly data processing pipeline (Steps 42–51).
Fig. 4: Example output of the micrograph analysis script.

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Acknowledgements

The Titan Krios microscopes were funded by the University of Leeds (UoL ABSL award) and the Wellcome Trust (108466/Z/15/Z). We are grateful to the EM community at Leeds and our external users for their feedback on our procedures and for the example data collection parameters shown in Table 1. We thank the Faculty of Biological sciences IT team at UoL, in particular P. Pelliccia, A. Richmond and M. Beck, for help with setting up and maintaining the data processing and storage servers and data transfer scripts. E.L.H. is partially funded by BBSRC (BB/L021250/1). M.G.I. received funding from the European Research Council (ERC) under the European Union’s Seventh Framework Programme (FP7/2007-2013) ERC grant agreement no. 322408 and the MRC (MR/P018491/1). The EPA_CC_threshold.py script is a modified version of a script kindly provided by R. Danev, who we also thank for helpful discussions about optimal use of the phase plate.

Author information

Authors and Affiliations

Authors

Contributions

R.F.T. and E.L.H. wrote the EPU setup protocol. M.G.I. and S.R. wrote the scripts. R.F.T., E.L.H., M.G.I., S.R. and N.A.R. contributed to the writing of the manuscript.

Corresponding authors

Correspondence to Rebecca F. Thompson or Neil A. Ranson.

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Competing interests

The authors declare no competing interests.

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Related links

Key references using this protocol

Hesketh, E. L. et al. Nat. Commun. 9, 2369 (2018): https://www.nature.com/articles/s41467-018-04793-6

Baggen, J. et al. Proc. Natl. Acad. Sci. USA 115, 397–402 (2018): http://www.pnas.org/content/115/2/397

Agip, A.-N. A. et al. Nat. Struct. Mol. Biol. 25, 548–556 (2018): https://www.nature.com/articles/s41594-018-0073-1

Integrated supplementary information

Supplementary Figure 1

Micrograph analysis output associated with example data (EMPAIR-10205).

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1, Supplementary Methods 1–3, Supplementary Notes 1 and 2, and Supplementary Tables 1–4

Reporting Summary

Supplementary Video 1

Steps 1–11 of the protocol, clipping grids for loading into a Thermo Fisher Scientific autoloader microscope

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Thompson, R.F., Iadanza, M.G., Hesketh, E.L. et al. Collection, pre-processing and on-the-fly analysis of data for high-resolution, single-particle cryo-electron microscopy. Nat Protoc 14, 100–118 (2019). https://doi.org/10.1038/s41596-018-0084-8

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