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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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.
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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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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