Journal: Nat Methods / Year: 2013 Title: Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Authors: Xueming Li / Paul Mooney / Shawn Zheng / Christopher R Booth / Michael B Braunfeld / Sander Gubbens / David A Agard / Yifan Cheng / 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 ...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.
History
Deposition
Mar 25, 2013
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Header (metadata) release
Apr 10, 2013
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Map release
May 1, 2013
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Update
Aug 26, 2015
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Current status
Aug 26, 2015
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK III
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Electron microscopy
Microscope
FEI POLARA 300
Details
Images were recorded in dose-fractionated format using K2 Summit operated in counting and super-resolution mode. Motion correction was performed for each image.
Date
Jun 1, 2012
Image recording
Category: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 600 / Average electron dose: 20 e/Å2
Tilt angle min
0
Tilt angle max
0
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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