Brief Communication

Achieving better-than-3-Å resolution by single-particle cryo-EM at 200 keV

  • Nature Methods volume 14, pages 10751078 (2017)
  • doi:10.1038/nmeth.4461
  • Download Citation
Received:
Accepted:
Published online:

Abstract

Nearly all single-particle cryo-EM structures resolved to better than 4-Å resolution have been determined using 300-keV transmission electron microscopes (TEMs). We demonstrate that it is possible to obtain reconstructions of macromolecular complexes of different sizes to better than 3-Å resolution using a 200-keV TEM. These structures are of sufficient quality to unambiguously assign amino acid rotameric conformations and identify ordered water molecules.

  • Subscribe to Nature Methods for full access:

    $59

    Subscribe

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

Accessions

Primary accessions

Electron Microscopy Data Bank

References

  1. 1.

    et al. Cell 165, 1698–1707 (2016).

  2. 2.

    , & eLife 6, e23006 (2017).

  3. 3.

    Q. Rev. Biophys. 28, 171–193 (1995).

  4. 4.

    et al. J. Struct. Biol. 188, 183–187 (2014).

  5. 5.

    et al. J. Mol. Biol. 429, 79–87 (2017).

  6. 6.

    , , , & J. Struct. Biol. 174, 1–10 (2011).

  7. 7.

    et al. J. Struct. Biol. 151, 41–60 (2005).

  8. 8.

    et al. J. Struct. Biol. 166, 95–102 (2009).

  9. 9.

    & Science 346, 1377–1380 (2014).

  10. 10.

    et al. Nat. Methods 14, 331–332 (2017).

  11. 11.

    & J. Struct. Biol. 192, 216–221 (2015).

  12. 12.

    J. Struct. Biol. 193, 1–12 (2016).

  13. 13.

    , , & eLife 5, e18722 (2016).

  14. 14.

    et al. Structure 20, 205–214 (2012).

  15. 15.

    , , , & eLife 4, e06380 (2015).

  16. 16.

    Ultramicroscopy 145, 85–93 (2014).

  17. 17.

    et al. Structure 20, 1823–1828 (2012).

  18. 18.

    et al. Nat. Methods 10, 584–590 (2013).

  19. 19.

    & J. Struct. Biol. 192, 188–195 (2015).

  20. 20.

    & eLife 4, e06980 (2015).

  21. 21.

    eLife 3, e03665 (2014).

  22. 22.

    , , & Cell 161, 438–449 (2015).

  23. 23.

    , , & Nat. Commun. 8, 16099 (2017).

  24. 24.

    et al. Nat. Methods 12, 943–946 (2015).

  25. 25.

    , & Setting up the Talos Arctica electron microscope and Gatan K2 direct detector for high-resolution cryogenic single-particle data acquisition. Protocol Exchange (2017).

  26. 26.

    et al. Q. Rev. Biophys. 21, 129–228 (1988).

  27. 27.

    , & Nat. Protoc. 3, 330–339 (2008).

  28. 28.

    & in Modern Electron Microscopy in Physical and Life Sciences. (eds. M. Janacek & R. Kral) (InTech, 2016).

  29. 29.

    , , , & J. Struct. Biol. 166, 205–213 (2009).

  30. 30.

    , & J. Struct. Biol. 143, 185–200 (2003).

  31. 31.

    J. Struct. Biol. 145, 91–99 (2004).

  32. 32.

    & Nat. Methods 9, 853–854 (2012).

  33. 33.

    et al. Ultramicroscopy 135, 24–35 (2013).

  34. 34.

    , , & Nat. Methods 14, 290–296 (2017).

  35. 35.

    , & J. Struct. Biol. 184, 226–236 (2013).

  36. 36.

    , & A multi-model approach to assessing local and global cryo-EM map quality. Preprint at bioRxiv (2017).

  37. 37.

    et al. Nucleic Acids Res. 32, D115–D119 (2004).

  38. 38.

    et al. eLife 5, e17219 (2016).

  39. 39.

    et al. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

  40. 40.

    et al. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

Download references

Acknowledgements

We thank J.-C. Ducom at The Scripps Research Institute High Performance Computing for computational support, B. Anderson at The Scripps Research Institute electron microscopy facility for microscope support, and M. Vos for advice and discussion regarding microscope alignments. We are grateful to Y. Cheng and Z. Yu (University of California, San Francisco) for kindly providing the 20S sample used in this study. M.A.H. is supported by a Helen Hay Whitney Foundation postdoctoral fellowship. G.C.L. is supported as a Searle Scholar and as a Pew Scholar, and by the US National Institutes of Health (NIH) grant DP2EB020402. Computational analyses of EM data were performed using shared instrumentation funded by NIH S10OD021634.

Author information

Author notes

    • Mark A Herzik Jr
    •  & Mengyu Wu

    These authors contributed equally to this work.

Affiliations

  1. Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA.

    • Mark A Herzik Jr
    • , Mengyu Wu
    •  & Gabriel C Lander

Authors

  1. Search for Mark A Herzik in:

  2. Search for Mengyu Wu in:

  3. Search for Gabriel C Lander in:

Contributions

M.A.H. and M.W. performed all cryo-EM experiments and analyses. M.A.H., M.W. and G.C.L. contributed to the experimental design and manuscript preparation.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Gabriel C Lander.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7, Supplementary Table 1 and Supplementary Notes 1–3

  2. 2.

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Protocol

    Setting up the Talos Arctica electron microscope and Gatan K2 direct detector for high-resolution cryogenic single-particle data acquisition