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

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TLDR
This approach 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 and greatly enhances image quality and data acquisition efficiency.
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 A). Using this approach, we determined a 3.3-A-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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Citations
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Structural Basis of Backwards Motion in Kinesin-1-Kinesin-14 Chimera: Implication for Kinesin-14 Motility

TL;DR: The C-terminal neck mimic plays a crucial role in coupling the chemical ATPase reaction with the mechanical cycle to produce the minus-end-directed motility of kinesin-14.
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Genome packaging of reovirus is mediated by the scaffolding property of the microtubule network.

TL;DR: It is demonstrated that the scaffolding properties of the microtubule, and not biochemical nature of tubulin, are critical determinants for reovirus efficient genome packaging.
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Electron cryo-tomography provides insight into procentriole architecture and assembly mechanism

TL;DR: The structure of the A-C linker that bridges neighboring triplets is described and two assembly intermediates at the growing ends of procentriole allowing us to propose a model for the pro centriole assembly.
Posted ContentDOI

Achieving Better Than 3 Å Resolution By Single Particle Cryo-EM At 200 keV

TL;DR: It is demonstrated that it is possible to obtain cryo-EM reconstructions of macromolecular complexes at a range of sizes to better than 3 Å resolution using a 200 keV TEM, of sufficient quality to unambiguously assign amino acid rotameric conformations and identify ordered water molecules.
Journal ArticleDOI

Higher-order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the Cvt vesicle

TL;DR: The findings suggest that Atg19 interaction limits Ape1 aggregate size while serving as a vehicle for vacuolar delivery of tetrameric Ams1 and key stages of Cvt vesicle biogenesis are visualize.
References
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Journal ArticleDOI

UCSF Chimera--a visualization system for exploratory research and analysis.

TL;DR: Two unusual extensions are presented: Multiscale, which adds the ability to visualize large‐scale molecular assemblies such as viral coats, and Collaboratory, which allows researchers to share a Chimera session interactively despite being at separate locales.
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Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy.

TL;DR: The hand determination and refinement optimization procedure is applied to image pairs of the dihydrolipoyl acetyltransferase (E2) catalytic core of the pyruvate dehydrogenase complex from Bacillus stearothermophilus taken by low-dose electron cryomicroscopy.
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Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 A resolution.

TL;DR: The three-dimensional structure of the proteasome from the archaebacterium Thermoplasma acidophilum has been elucidated by x-ray crystallographic analysis by means of isomorphous replacement and cyclic averaging.
Journal ArticleDOI

Accurate determination of local defocus and specimen tilt in electron microscopy

TL;DR: Two computer programs are presented, CTFFIND3 and CTFTILT, which determine defocus parameters from images of untilted specimens, as well as defocus and tilt parameters from image of tilted specimens, respectively, using a simple algorithm.
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Prevention of overfitting in cryo-EM structure determination

TL;DR: Analysis of simulated data with realistic signal-to-noise ratios indicates that the accuracy of the orientation determination is not affected by the exclusion of high-frequency terms, nor by the use of a model that is reconstructed from only half of the particles, as expected.
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