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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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Antibiotic export by MexB multidrug efflux transporter is allosterically controlled by a MexA-OprM chaperone-like complex.

TL;DR: It is unveiled that MexB inserted in lipid membrane is not set for active transport because it displays an inactive LTC form with a Closed exit gate, and shed new light on the resistance nodulation cell division (RND) cognate partners which act as allosteric factors eliciting the functional drug extrusion.
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Structural insights into ribosomal rescue by Dom34 and Hbs1 at near-atomic resolution.

TL;DR: The structure of Dom34 and Hbs1 bound to a yeast ribosome programmed with a nonstop mRNA at 3.3 Å resolution is solved using cryo-electron microscopy and establishes the structural foundation for the recognition of aberrantly stalled 80S ribosomes by the Dom34·Hbs1·GTP complex during Dom34-mediated mRNA surveillance pathways.
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Tc toxin activation requires unfolding and refolding of a β-propeller

TL;DR: This work shows in atomic detail how the assembly of the three components activates the toxin, and finds that part of the cocoon completely unfolds and refolds into an alternative conformation upon binding.
Journal ArticleDOI

Structural assembly of the tailed bacteriophage ϕ29.

TL;DR: Near-atomic structures of the ϕ29 pre-genome packaging head (prohead), the mature virion and the genome-emptied virion are reported, providing insights into DNA packaging and release.
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Ribosome-stalk biogenesis is coupled with recruitment of nuclear-export factor to the nascent 60S subunit

TL;DR: A spatiotemporal mark on the ribosomal stalk controls the recruitment of an RNA-export receptor to the nascent 60S subunit, facilitating nuclear export of the large subunit.
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.
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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