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. 2013 Jun;10(6):584-90.
doi: 10.1038/nmeth.2472. Epub 2013 May 5.

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

Xueming Li  1 , Paul Mooney, Shawn Zheng, Christopher R Booth, Michael B Braunfeld, Sander Gubbens, David A Agard, Yifan Cheng
Affiliations

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

Xueming Li et al. Nat Methods. 2013 Jun.

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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Conflict of interest statement

COMPETING FINANCIAL INTEREST

The authors declare competing financial interests: details are available in the online version of the paper.

Figures

Figure 1
Figure 1. Detective Quantum Efficiency (DQE) and Detector Conversion Efficiency (DCE) of K2 Summit electron counting camera
a. DQEs of K2 Summit were measured in both counting (red curve, taken at 2.36 e−/pixel/sec) and linear charge accumulation modes (blue curve), and are compared with the DQE of a typical scintillator-based CCD camera, the Gatan US4000 Ultrascan (black). b. The dose dependent DCEs of K2 Summit counting mode. Electron counts were measured as a function of incident electron dose rates and were fit to a polynomial curve. The straight line represents the ideal linear response with a slope of 0.87, corresponding to the quantum efficiency of the camera.
Figure 2
Figure 2. Motion correction restores the lost high-resolution information
a. Fourier transforms of an image of frozen hydrated archaeal 20S proteasomes. This is a near “perfect” image where Thon rings extend to near 3 Å. The cross correlation (CC) between image Thon rings at 10 ~ 5Å and simulated ideal Thon rings over the same resolution range is 0.192. b. The 24 individual subframes were cross correlated and relative positional shifts determined as described in the text and Supplementary Methods. Based on these calculations, the path of motion between the first subframe (large black dot) and last can be determined. c. Fourier transform of the same image after motion correction, where the Thon ring CC is now 0.233. d. Fourier transform of a worse image showing a predominantly unidirectional resolution cut-off at ~20Å. The Thon ring CC is only 0.092. e. The trace of motion between subframes. f. Fourier transform of the same image after motion correction showing that resolution has been isotropically restored, and the Thon ring CC improved to 0.238. The narrow white band was caused by residual fixed pattern noise in each frame, which was subsequently eliminated.
Figure 3
Figure 3. Analysis of motion induced image blurring on resolution of the 3D reconstruction
a. Comparison of Fourier Shell Correlation (FSC) curves from 3D reconstructions using images without motion correction (red), images corrected using the entire subframe and containing all subframes (purple), and images corrected by subregion and containing subframe 3 – 15 (blue). Horizontal dashed lines are shown for both the FSC = 0.5 and 0.143 criteria. The FSC curve between the final map and that calculated from the fitted atomic model is shown in gold. b. Comparison of rotational averages of Fourier power spectra of the different 3D reconstructions (the same colors as in a). c. The average speed of motion from the entire dataset of 553 images vs. subframe number is shown (blue). Error bars indicate standard deviations of each average. Note the roughly exponential decay of initial motion as the sample equilibrates in the electron beam. The quality of 3D reconstructions calculated using particle data taken from the indicated individual subframes are quantified by the FSC value at 5 Å resolution (red) derived from Supplementary Fig. 5a. The same particle parameters were used for all 24 3D reconstructions.
Figure 4
Figure 4. Subregion motion correction
a. An image of vitreous ice embedded 20S proteasomes taken completely within a single hole. Red and blue squares represent two different subregions of 2048 × 2048 pixels. b. Comparison of displacement traces determined from the two subregions (black and blue) with that determined using the entire subframe (red). Note that the trends of motions in the subregions are similar, although their speeds in the first few subframes are different. c. Subframe displacement traces from the same image after removing the first 5 frames. The difference between two regions is significantly less than that in b. d–f. Similar to a–c, but the image spans both the hole and the surrounding supporting carbon film. The displacement traces from subregions of carbon film and vitreous ice indicate that they behave very differently under the electron beam. After removal of the first 5 subframes, the remaining shifts are similarly reduced.
Figure 5
Figure 5
Final 3D reconstruction of archaeal 20S proteasome reveals clear side chain detail a. 3D density map of T. acidophilum 20S proteasome filtered to a resolution of 3.3 Å. b. Two different views of asymmetrical α- and β-subunit segmented from the 3D density map shown in a. The main chain can be traced throughout the entire map. c. Two α-helices segmented from the α-and β-subunits showing clear density for the majority of side chains. d. A portion of the cryoEM density map showing clear side chain densities. The docked atomic structure was refined to fit the density map by molecular dynamic flexible fitting procedure. e. The same portion of a 2Fo-Fc map of 3.4 Å crystal structure calculated using the atomic structure (pdb code: 1PMA).
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