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

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.

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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.

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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.

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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.

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Correspondence to David A Agard or Yifan Cheng.

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

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Motion correction for dose fractionation (ZIP 502 kb)

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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

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