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Open AccessJournal ArticleDOI

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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BookDOI

Membrane Protein Complexes:: Structure and Function

TL;DR: This edited book is aimed at the advanced undergraduates, postgraduates and established researchers within this broad field of study and is hoped that the book will be of interest and use to those involved with the study of cellular membranes and their associated proteins.
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Cellular electron cryo tomography and in situ sub-volume averaging reveal the context of microtubule-based processes

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Journal ArticleDOI

Conformational Landscape of the p28-Bound Human Proteasome Regulatory Particle.

TL;DR: Cryogenic electron microscopy analysis suggests that p28 assists the proteolytic core particle to select a specific conformation of the ATPase ring for RP engagement and is released in a shoehorn-like fashion in the last step of the chaperone-mediated proteasome assembly.
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.
Journal ArticleDOI

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.
Journal ArticleDOI

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.
Journal ArticleDOI

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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