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


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.


  1. Cheng, Y., Grigorieff, N., Penczek, P. A. & Walz, T. A primer to single-particle cryo-electron microscopy. Cell 161, 438–449 (2015).

    Article  CAS  Google Scholar 

  2. Renaud, J.-P. et al. Cryo-EM in drug discovery: achievements, limitations and prospects. Nat. Rev. Drug Discov. 17, 471–492 (2018).

    Article  CAS  Google Scholar 

  3. Thompson, R. F., Walker, M., Siebert, C. A., Muench, S. P. & Ranson, N. A. An introduction to sample preparation and imaging by cryo-electron microscopy for structural biology. Methods 100, 3–15 (2016).

    Article  CAS  Google Scholar 

  4. Kuehlbrandt, W. The resolution revolution. Science 343, 1443–1444 (2014).

    Article  Google Scholar 

  5. Ruskin, R. S., Yu, Z. & Grigorieff, N. Quantitative characterization of electron detectors for transmission electron microscopy. J. Struct. Biol. 184, 385–393 (2013).

    Article  CAS  Google Scholar 

  6. Kuijper, M. et al. FEI’s direct electron detector developments: embarking on a revolution in cryo-TEM. J. Struct. Biol. 192, 179–187 (2015).

    Article  Google Scholar 

  7. McMullan, G., Faruqi, A. R., Clare, D. & Henderson, R. Comparison of optimal performance at 300 keV of three direct electron detectors for use in low dose electron microscopy. Ultramicroscopy 147, 156–163 (2014).

    Article  CAS  Google Scholar 

  8. Patwardhan, A. Trends in the Electron Microscopy Data Bank (EMDB). Acta Crystallogr. D Struct. Biol. 73, 503–508 (2017).

    Article  CAS  Google Scholar 

  9. Noble, A. J. et al. Routine single particle CryoEM sample and grid characterization by tomography. Elife 7, 32 (2018).

    Google Scholar 

  10. Drulyte, I. et al. Approaches to altering particle distributions in cryo-electron microscopy sample preparation. Acta Crystallogr. D Struct. Biol. 74, 560–571 (2018).

    Article  CAS  Google Scholar 

  11. Amporndanai, K. et al. X-ray and cryo-EM structures of inhibitor-bound cytochrome bc1 complexes for structure-based drug discovery. IUCrJ. 5, 200–210 (2018).

    Article  CAS  Google Scholar 

  12. Meshcheriakova, Y., Durrant, A., Hesketh, E. L., Ranson, N. A. & Lomonossoff, G. P. Combining high-resolution cryo-electron microscopy and mutagenesis to develop cowpea mosaic virus for bionanotechnology. Biochem. Soc. Trans. 45, 1263–1269 (2017).

    Article  CAS  Google Scholar 

  13. Rawson, S. et al. Elucidating the structural basis for differing enzyme inhibitor potency by cryo-EM. Proc. Natl. Acad. Sci.USA 115, 1795–1800 (2018).

    Article  CAS  Google Scholar 

  14. Baggen, J. et al. Role of enhanced receptor engagement in the evolution of a pandemic acute hemorrhagic conjunctivitis virus. Proc. Natl. Acad. Sci.USA 115, 397–402 (2018).

    Article  CAS  Google Scholar 

  15. Scheres, S. H. W. A Bayesian view on cryo-EM structure determination. J. Mol. Biol. 415, 406–418 (2012).

    Article  CAS  Google Scholar 

  16. Fernandez-Leiro, R. & Scheres, S. H. W. A pipeline approach to single-particle processing in RELION. Acta Crystallogr. D Struct. Biol. 73, 496–502 (2017).

    Article  CAS  Google Scholar 

  17. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  Google Scholar 

  18. Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    Article  CAS  Google Scholar 

  19. Tan, Y. Z. et al. Addressing preferred specimen orientation in single-particle cryo-EM through tilting. Nat. Methods 14, 793–796 (2017).

    Article  CAS  Google Scholar 

  20. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  Google Scholar 

  21. Suloway, C. et al. Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 151, 41–60 (2005).

    Article  CAS  Google Scholar 

  22. la Rosa-Trevin de, J. M. et al. Scipion: a software framework toward integration, reproducibility and validation in 3D electron microscopy. J. Struct. Biol. 195, 93–99 (2016).

    Article  Google Scholar 

  23. Biyani, N. et al. Focus: the interface between data collection and data processing in cryo-EM. J. Struct. Biol. 198, 124–133 (2017).

    Article  CAS  Google Scholar 

  24. Grant, T. & Grigorieff, N. Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6. Elife 4, e06980 (2015).

    Article  Google Scholar 

  25. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  Google Scholar 

  26. Russo, C. J. & Passmore, L. A. Electron microscopy. Ultrastable gold substrates for electron cryomicroscopy. Science 346, 1377–1380 (2014).

    Article  CAS  Google Scholar 

  27. Hesketh, E. L. et al. The 3.3 Å structure of a plant geminivirus using cryo-EM. Nat. Commun. 9, 2369 (2018).

    Article  Google Scholar 

  28. Iadanza, M. G. et al. The structure of a β2-microglobulin fibril suggests a molecular basis for its amyloid polymorphism. Nat. Commun. 9, 4517 (2018).

    Article  Google Scholar 

  29. Hurdiss, D. L., Frank, M., Snowden, J. S., Macdonald, A. & Ranson, N. A. The structure of an infectious human polyomavirus and its interactions with cellular receptors. Structure 26, 839–847.e3 (2018).

    Article  CAS  Google Scholar 

  30. Plaschka, C., Lin, P.-C., Charenton, C. & Nagai, K. Prespliceosome structure provides insights into spliceosome assembly and regulation. Nature 559, 419–422 (2018).

    Article  CAS  Google Scholar 

  31. Conley, M. J. et al. Calicivirus VP2 forms a portal to mediate endosome escape. Preprint at (2018).

  32. Danev, R., Buijsse, B., Khoshouei, M., Plitzko, J. M. & Baumeister, W. Volta potential phase plate for in-focus phase contrast transmission electron microscopy. Proc. Natl. Acad. Sci. 111, 15635–15640 (2014).

    Article  CAS  Google Scholar 

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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 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.

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Authors and Affiliations



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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The authors declare no competing interests.

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Key references using this protocol

Hesketh, E. L. et al. Nat. Commun. 9, 2369 (2018):

Baggen, J. et al. Proc. Natl. Acad. Sci. USA 115, 397–402 (2018):

Agip, A.-N. A. et al. Nat. Struct. Mol. Biol. 25, 548–556 (2018):

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).

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