Nothing Special   »   [go: up one dir, main page]
Skip to main content

Thank you for visiting nature.com. 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.

  • Article
  • Published:

Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM

Nature Methods volume 10, pages 584–590 (2013)Cite this article

Subjects

Abstract

In recent work with large high-symmetry viruses, single-particle electron cryomicroscopy (cryo-EM) has achieved the determination of near-atomic-resolution structures by allowing direct fitting of atomic models into experimental density maps. However, achieving this goal with smaller particles of lower symmetry remains challenging. Using a newly developed single electron–counting detector, we confirmed that electron beam–induced motion substantially degrades resolution, and we showed that the combination of rapid readout and nearly noiseless electron counting allow image blurring to be corrected to subpixel accuracy, restoring intrinsic image information to high resolution (Thon rings visible to ∼3 Å). Using this approach, we determined a 3.3-Å-resolution structure of an ∼700-kDa protein with D7 symmetry, the Thermoplasma acidophilum 20S proteasome, showing clear side-chain density. Our method greatly enhances image quality and data acquisition efficiency—key bottlenecks in applying near-atomic-resolution cryo-EM to a broad range of protein samples.

Access through your institution
Buy or subscribe

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

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Figure 1: Detective quantum efficiency (DQE) and detector conversion efficiency (DCE) of the K2 Summit electron-counting camera.
Figure 2: Motion correction restores the lost high-resolution information.
Figure 3: Analysis of motion-induced image blurring on resolution of the 3D reconstruction.
Figure 4: Subregion motion correction.
Figure 5: Final 3D reconstruction of the T. acidophilum archaeal 20S proteasome.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Wolf, M., Garcea, R.L., Grigorieff, N. & Harrison, S.C. Subunit interactions in bovine papillomavirus. Proc. Natl. Acad. Sci. USA 107, 6298–6303 (2010)10.1073/pnas.0914604107.

    Article  PubMed  PubMed Central  Google Scholar 

  2. 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  PubMed  PubMed Central  Google Scholar 

  3. Chen, J.Z. et al. Molecular interactions in rotavirus assembly and uncoating seen by high-resolution cryo-EM. Proc. Natl. Acad. Sci. USA 106, 10644–10648 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Zhang, X. et al. Near-atomic resolution using electron cryomicroscopy and single-particle reconstruction. Proc. Natl. Acad. Sci. USA 105, 1867–1872 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Zhang, R. et al. 4.4 Å cryo-EM structure of an enveloped alphavirus Venezuelan equine encephalitis virus. EMBO J. 30, 3854–3863 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Zhang, J. et al. Mechanism of folding chamber closure in a group II chaperonin. Nature 463, 379–383 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 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  PubMed  Google Scholar 

  8. McMullan, G., Chen, S., Henderson, R. & Faruqi, A.R. Detective quantum efficiency of electron area detectors in electron microscopy. Ultramicroscopy 109, 1126–1143 (2009)10.1016/j.ultramic.2009.04.002.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. McMullan, G. et al. Experimental observation of the improvement in MTF from backthinning a CMOS direct electron detector. Ultramicroscopy 109, 1144–1147 (2009)10.1016/j.ultramic.2009.05.005.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. McMullan, G., Clark, A.T., Turchetta, R. & Faruqi, A.R. Enhanced imaging in low dose electron microscopy using electron counting. Ultramicroscopy 109, 1411–1416 (2009)10.1016/j.ultramic.2010.07.012.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Bammes, B.E., Rochat, R.H., Jakana, J., Chen, D.H. & Chiu, W. Direct electron detection yields cryo-EM reconstructions at resolutions beyond 3/4 Nyquist frequency. J. Struct. Biol. 177, 589–601 (2012)10.1016/j.jsb.2012.01.008.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Milazzo, A.C. et al. Initial evaluation of a direct detection device detector for single particle cryo-electron microscopy. J. Struct. Biol. 176, 404–408 (2011)10.1016/j.jsb.2011.09.002.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Henderson, R. & Glaeser, R.M. Quantitative analysis of image contrast in electron micrographs of beam-sensitive crystals. Ultramicroscopy 16, 139–150 (1985).

    Article  CAS  Google Scholar 

  14. Glaeser, R.M., McMullan, G., Faruqi, A.R. & Henderson, R. Images of paraffin monolayer crystals with perfect contrast: minimization of beam-induced specimen motion. Ultramicroscopy 111, 90–100 (2011)10.1016/j.ultramic.2010.10.010.

    Article  CAS  PubMed  Google Scholar 

  15. Typke, D., Gilpin, C.J., Downing, K.H. & Glaeser, R.M. Stroboscopic image capture: reducing the dose per frame by a factor of 30 does not prevent beam-induced specimen movement in paraffin. Ultramicroscopy 107, 106–115 (2007)10.1016/j.ultramic.2006.06.005.

    Article  CAS  PubMed  Google Scholar 

  16. Miyazawa, A., Fujiyoshi, Y., Stowell, M. & Unwin, N. Nicotinic acetylcholine receptor at 4.6 Å resolution: transverse tunnels in the channel wall. J. Mol. Biol. 288, 765–786 (1999).

    Article  CAS  PubMed  Google Scholar 

  17. Jensen, G.J. Alignment error envelopes for single particle analysis. J. Struct. Biol. 133, 143–155 (2001)10.1006/jsbi.2001.4334.

    Article  CAS  PubMed  Google Scholar 

  18. Rosenthal, P.B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).

    Article  CAS  PubMed  Google Scholar 

  19. Saad, A. et al. Fourier amplitude decay of electron cryomicroscopic images of single particles and effects on structure determination. J. Struct. Biol. 133, 32–42 (2001).

    Article  CAS  PubMed  Google Scholar 

  20. Brilot, A.F. et al. Beam-induced motion of vitrified specimen on holey carbon film. J. Struct. Biol. 177, 630–637 (2012)10.1016/j.jsb.2012.02.003.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Campbell, M.G. et al. Movies of ice-embedded particles enhance resolution in electron cryo-microscopy. Structure 20, 1823–1828 (2012)10.1016/j.str.2012.08.026.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 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)10.7554/eLife.00461.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Battaglia, M. et al. A rad-hard CMOS active pixel sensor for electron microscopy. Nucl. Instrum. Methods Phys. Res. A 598, 642–649 (2009)10.1016/j.nima.2008.09.029.

    Article  CAS  Google Scholar 

  24. Bichsel, H. Straggling in thin silicon detectors. Rev. Mod. Phys. 60, 663–699 (1988)10.1103/RevModPhys.60.663.

    Article  CAS  Google Scholar 

  25. Booth, C.R., Jakana, J. & Chiu, W. Assessing the capabilities of a 4kx4k CCD camera for electron cryo-microscopy at 300 kV. J. Struct. Biol. 156, 556–563 (2006)10.1016/j.jsb.2006.08.019.

    Article  CAS  PubMed  Google Scholar 

  26. Löwe, J. et al. Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 Å resolution. Science 268, 533–539 (1995).

    Article  PubMed  Google Scholar 

  27. Rabl, J. et al. Mechanism of gate opening in the 20S proteasome by the proteasomal ATPases. Mol. Cell 30, 360–368 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Mindell, J.A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003).

    Article  PubMed  Google Scholar 

  29. Scheres, S.H. & Chen, S. Prevention of overfitting in cryo-EM structure determination. Nat. Methods 9, 853–854 (2012)10.1038/nmeth.2115.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Yu, Y. et al. Interactions of PAN's C-termini with archaeal 20S proteasome and implications for the eukaryotic proteasome-ATPase interactions. EMBO J. 29, 692–702 (2010).

    Article  CAS  PubMed  Google Scholar 

  31. Stark, H., Zemlin, F. & Boettcher, C. Electron radiation damage to protein crystals of bacteriorhodopsin at different temperatures. Ultramicroscopy 63, 75–79 (1996)10.1016/0304-3991(96)00045-9.

    Article  CAS  Google Scholar 

  32. Mooney, P. Optimization of image collection for cellular electron microscopy. Methods Cell Biol. 79, 661–719 (2007)10.1016/S0091-679X(06)79027-6.

    Article  PubMed  Google Scholar 

  33. Meyer, R.R., Kirkland, A.I., Dunin-Borkowski, R.E. & Hutchison, J.L. Experimental characterisation of CCD cameras for HREM at 300 kV. Ultramicroscopy 85, 9–13 (2000).

    Article  CAS  PubMed  Google Scholar 

  34. Roseman, A.M. FindEM–a fast, efficient program for automatic selection of particles from electron micrographs. J. Struct. Biol. 145, 91–99 (2004).

    Article  CAS  PubMed  Google Scholar 

  35. 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  PubMed  PubMed Central  Google Scholar 

  36. Li, X., Grigorieff, N. & Cheng, Y. GPU-enabled FREALIGN: accelerating single particle 3D reconstruction and refinement in Fourier space on graphics processors. J. Struct. Biol. 172, 407–412 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Pettersen, E.F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  38. 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  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank K. Egami (UCSF) for purifying T. acidophilum 20S proteasome. We thank B. Lee for support in system optimization and DQE analysis and T. Sha for support in integrating the camera into the UCSF software environment. M. Lent was a principal architect of the camera and supported testing and troubleshooting of our prototype camera. This work is supported by the HHMI (D.A.A.) and US National Science Foundation grant DBI-0960271 to D.A.A and Y.C., which in part funded the development of the K2 camera in association with Gatan and P. Denes at Lawrence Berkeley Labs. An initial grant from the HHMI funded the first pixel prototype chip in collaboration with P. Denes. This work is also supported by the UCSF Program for Breakthrough Biomedical Research and US National Institutes of Health grants R01GM082893, R01GM098672 and S10RR026814 to Y.C. and P50GM082250 to A. Frankel.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, The Keck Advanced Microscopy Laboratory, University of California, San Francisco (UCSF), San Francisco, California, USA

    Xueming Li, Shawn Zheng, Michael B Braunfeld, David A Agard & Yifan Cheng

  2. Gatan Inc., Pleasanton, California, USA

    Paul Mooney, Christopher R Booth & Sander Gubbens

  3. The Howard Hughes Medical Institute (HHMI), UCSF, San Francisco, California, USA

    Shawn Zheng, Michael B Braunfeld & David A Agard

Authors
  1. Xueming Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Paul Mooney
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shawn Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Christopher R Booth
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael B Braunfeld
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sander Gubbens
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. David A Agard
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yifan Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

X.L., D.A.A. and Y.C. designed experiments. X.L. carried out all experiments. P.M. and C.K.B. determined DQE curves (Fig. 1a). S.Z. participated in implementing the K2 and dose fractionation. S.G. was the chief architect of the K2 project and, along with P.M., contributed significant scientific and technical insights throughout the project. All of the data in Figure 1a were collected at UCSF, and all of the other figures are based solely on experiments carried in the laboratories of Y.C. and D.A.A. M.B.B. provided technical assistance in operating the microscope. X.L., D.A.A. and Y.C. wrote the manuscript. All authors participated in discussion and revision of the manuscript.

Corresponding authors

Correspondence to David A Agard or Yifan Cheng.

Ethics declarations

Competing interests

C.K.B., P.M. and S.G. are employees of Gatan Inc., which developed and is marketing the K2 camera.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 (PDF 8693 kb)

Supplementary Software

Motion correction for dose fractionation (ZIP 502 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Li, X., Mooney, P., Zheng, S. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat Methods 10, 584–590 (2013). https://doi.org/10.1038/nmeth.2472

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.2472

This article is cited by

Search

Advanced search

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