Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM
Xueming Li,Paul Mooney,Shawn Q. Zheng,Shawn Q. Zheng,Christopher R. Booth,Michael B. Braunfeld,Michael B. Braunfeld,Sander Gubbens,David A. Agard,David A. Agard,Yifan Cheng +10 more
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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.read more
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In Situ Structures of the Polymerase Complex and RNA Genome Show How Aquareovirus Transcription Machineries Respond to Uncoating.
Ke Ding,L. Nguyen,Z. H. Zhou +2 more
TL;DR: In this article, the in situ structures of the RNA-dependent RNA polymerase (RdRp) and NTPase are known for the single-layer reovirus cytoplasmic polyhedrosis virus (CPV), but not for multilayered reoviruses (ARV), which possess a primed stage that CPV lacks.
Journal ArticleDOI
Practical considerations for using K3 cameras in CDS mode for high-resolution and high-throughput single particle cryo-EM.
Ming Sun,Caleigh M. Azumaya,Eric Tse,David Bulkley,Matthew B. Harrington,Glenn Gilbert,Adam Frost,Daniel R. Southworth,Kliment A. Verba,Yifan Cheng,David A. Agard +10 more
TL;DR: In this paper, the Gatan K3 was used for protein structure determination and achieved high-resolution and high-throughput reconstruction of heavy-chain apoferritin protein complexes.
Journal ArticleDOI
3D reconstruction of two-dimensional crystals
TL;DR: 2D electron crystallography is particularly attractive for the structural analysis of membrane proteins that are too small for single particle analyses and too unstable to form 3D crystals.
Journal ArticleDOI
Inhibition of calcium-triggered secretion by hydrocarbon-stapled peptides
Ying Lai,Giorgio Fois,J. Reyes Flores,Michael J. Tuvim,Qiangjun Zhou,Kailu Yang,Jeremy Leitz,John Peters,Yunxiang Zhang,Richard A. Pfuetzner,Luis Esquivies,Philip H. Jones,Manfred Frick,Burton F. Dickey,Axel T. Brunger +14 more
TL;DR: In this article , a hydrocarbon-stapled peptide was designed to specifically disrupt Ca2+-triggered membrane fusion by interfering with the so-called primary interface between the neuronal SNARE complex and the Ca2-binding C2B domain of synaptotagmin-1.
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Mechanistic insights into the SNARE complex disassembly.
Xuan Huang,Shan Sun,Xiaojing Wang,Fenghui Fan,Qiang Zhou,Shan Lu,Yong Cao,Qiu-Wen Wang,Meng-Qiu Dong,Jun Yao,Sen-Fang Sui +10 more
TL;DR: The studies provide a rotation model of α-SNAP–mediated disassembly of the SNARE complex and define the interaction between the amino terminus of theSNARE helical bundle and the pore loop of the NSF-D1 domain and demonstrate its essential role as a potential anchor forSNARE complex disassembly.
References
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Prevention of overfitting in cryo-EM structure determination
Sjors H.W. Scheres,Shaoxia Chen +1 more
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