Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Advances in the field of single-particle cryo-electron microscopy over the last decade


In single-particle cryo-electron microscopy (cryo-EM), molecules suspended in a thin aqueous layer are rapidly frozen and imaged at cryogenic temperature in the transmission electron microscope. From the random projection views, a three-dimensional image is reconstructed, enabling the structure of the molecule to be obtained. In this article I discuss technological progress over the past decade, which has, in my own field of study, culminated in the determination of ribosome structure at 2.5-Å resolution. I also discuss likely future improvements in methodology.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type



Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2: Improvements in resolution of the ribosome structure over the past decade.


  1. Grassucci, R.A., Taylor, D.J. & Frank, J. Preparation of macromolecular complexes for cryo-electron microscopy. Nat. Protoc. 2, 3239–3246 (2007).

    Article  CAS  Google Scholar 

  2. Grassucci, R.A., Taylor, D. & Frank, J. Visualization of macromolecular complexes using cryo-electron microscopy with FEI Tecnai transmission electron microscopes. Nat. Protoc. 3, 330–339 (2008).

    Article  CAS  Google Scholar 

  3. Shaikh, T.R. et al. SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs. Nat. Protoc. 3, 1941–1974 (2008).

    Article  CAS  Google Scholar 

  4. Trabuco, L.G., Villa, E., Mitra, K., Frank, J. & Schulten, K. Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics. Structure 16, 673–683 (2008).

    Article  CAS  Google Scholar 

  5. Scheres, S.H.W. et al. Disentangling conformational states of macromolecules in 3D-EM through likelihood optimization. Nat. Methods 4, 27–29 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  7. Zhang, X., Jin, L., Fang, Q., Hui, W.H. & Zhou, Z.H. 3.3 A cryo-EM structure of a nonenveloped virus reveals a priming mechanism for cell entry. Cell 141, 472–482 (2010).

    Article  CAS  Google Scholar 

  8. LeBarron, J. et al. Exploration of parameters in cryo-EM leading to an improved density map of the E. coli ribosome. J. Struct. Biol. 164, 24–32 (2008).

    Article  CAS  Google Scholar 

  9. Villa, E. et al. Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis. Proc. Natl. Acad. Sci. USA 106, 1063–1068 (2009).

    Article  CAS  Google Scholar 

  10. Hashem, Y. et al. High-resolution cryo-electron microscopy structure of the Trypanosoma brucei ribosome. Nature 494, 385–389 (2013).

    Article  CAS  Google Scholar 

  11. Henderson, R. The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules. Q. Rev. Biophys. 28, 171–193 (1995).

    Article  CAS  Google Scholar 

  12. Frank, J. et al. A model of protein synthesis based on cryo-electron microscopy of the E. coli ribosome. Nature 376, 441–444 (1995).

    Article  CAS  Google Scholar 

  13. Bai, X.C., Fernandez, I.S., McMullan, G. & Scheres, S.H.W. Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles. eLife 2, e00461 (2013).

    Article  Google Scholar 

  14. Liu, Z. et al. Structure and assembly model for the Trypanosoma cruzi 60S ribosomal subunit. Proc. Natl. Acad. Sci. USA 113, 12174–12179 (2016).

    Article  CAS  Google Scholar 

  15. Liao, M., Cao, E., Julius, D. & Cheng, Y. Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature 504, 107–112 (2013).

    Article  CAS  Google Scholar 

  16. Cao, E., Liao, M., Cheng, Y. & Julius, D. TRPV1 structures in distinct conformations reveal activation mechanisms. Nature 504, 113–118 (2013).

    Article  CAS  Google Scholar 

  17. Jain, T., Sheehan, P., Crum, J., Carragher, B. & Potter, C.S. Spotiton: a prototype for an integrated inkjet dispense and vitrification system for cryo-TEM. J. Struct. Biol. 179, 68–75 (2012).

    Article  Google Scholar 

  18. Razinkov, I. et al. A new method for vitrifying samples for cryoEM. J. Struct. Biol. 195, 190–198 (2016).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  20. Russo, C.J. & Passmore, L.A. Progress towards an optimal specimen support for electron cryomicroscopy. Curr. Opin. Struct. Biol. 37, 81–89 (2016).

    Article  CAS  Google Scholar 

  21. Crowther, R.A. (ed.) The Resolution Revolution: Recent Advances in CryoEM (Methods in Enzymology Volume 579)(Academic Press/Elsevier, 2016).

  22. Danev, R. & Baumeister, W. Cryo-EM single particle analysis with the Volta phase plate. eLife 5, e13046 (2016).

    Article  Google Scholar 

  23. Lu, Z. et al. Monolithic microfluidic mixing-spraying devices for time-resolved cryo-electron microscopy. J. Struct. Biol. 168, 388–395 (2009).

    Article  Google Scholar 

  24. Chen, B. et al. Structural dynamics of ribosome subunit association studied by mixing-spraying time-resolved cryogenic electron microscopy. Structure 23, 1097–1105 (2015).

    Article  CAS  Google Scholar 

  25. Fu, Z. et al. Key intermediates in ribosome recycling visualized by time-resolved cryoelectron microscopy. Structure 24, 2092–2101 (2016).

    Article  CAS  Google Scholar 

  26. Bai, X., Rajendra, E., Yang, G., Shi, Y. & Scheres, S.H.W. Sampling the conformational space of the catalytic subunit of human g-secretase. eLife 4, e11182 (2015).

    Article  Google Scholar 

  27. Liu, Z. et al. Determination of the ribosome structure to a resolution of 2.5 Å by single-particle cryo-EM. Protein Sci. (2016).

  28. Schüler, M. et al. Structure of the ribosome-bound cricket paralysis virus IRES RNA. Nat. Struct. Mol. Biol. 13, 1092–1096 (2006).

    Article  Google Scholar 

  29. Armache, J.P. et al. Cryo-EM structure and rRNA model of a translating eukaryotic 80S ribosome at 5.5-A resolution. Proc. Natl. Acad. Sci. USA 107, 19748–19753 (2010).

    Article  CAS  Google Scholar 

  30. Wong, W. et al. Cryo-EM structure of the Plasmodium falciparum 80S ribosome bound to the anti-protozoan drug emetine. eLife 3, e03080 (2014).

    Article  Google Scholar 

Download references


This work has been supported by the Howard Hughes Medical Institute and by US National Institutes of Health grant R01 GM29169.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Joachim Frank.

Ethics declarations

Competing interests

The author declares no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Frank, J. Advances in the field of single-particle cryo-electron microscopy over the last decade. Nat Protoc 12, 209–212 (2017).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing