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
Citations
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Gene Expression Is Circular: Factors for mRNA Degradation Also Foster mRNA Synthesis
Gal Haimovich,Daniel A. Medina,Sebastien Causse,Manuel Garber,Gonzalo Millán-Zambrano,Oren Barkai,Sebastián Chávez,José E. Pérez-Ortín,Xavier Darzacq,Mordechai Choder +9 more
TL;DR: It is demonstrated that most yeast mRNAs are degraded by the cytoplasmic 5'-to-3' pathway (the "decaysome"), as proposed previously, and the level of these m RNAs is highly robust to perturbations in this major pathway because defects in various decaysome components lead to transcription downregulation.
Journal ArticleDOI
Comparison of optimal performance at 300keV of three direct electron detectors for use in low dose electron microscopy
TL;DR: The K2 Summit has the best D QE at low spatial frequencies but with increasing spatial frequency its DQE falls below that of the Falcon II and the FEI Falcon II, and it is found that all three detectors have a betterDQE than film.
Journal ArticleDOI
Atomic-accuracy models from 4.5-A cryo-electron microscopy data with density-guided iterative local refinement
Frank DiMaio,Yifan Song,Xueming Li,Matthias J. Brunner,Chunfu Xu,Vincent P. Conticello,Edward H. Egelman,Thomas C. Marlovits,Yifan Cheng,David Baker +9 more
TL;DR: A general approach for refining protein structure models on the basis of cryo-electron microscopy maps with near-atomic resolution that integrates Monte Carlo sampling with local density-guided optimization, Rosetta all-atom refinement and real-space B-factor fitting is described.
Journal ArticleDOI
Structure of a yeast spliceosome at 3.6-angstrom resolution
TL;DR: The near-atomic-resolution structure of a central spliceosome provides a molecular framework for mechanistic understanding of pre-mRNA splicing and reveals insights into the structure and function of the yeast splicesome.
Journal ArticleDOI
Structure of the Mammalian Ribosome-Sec61 Complex to 3.4 Å Resolution
TL;DR: Comparison of the maps from idle and translating complexes suggests how conformational changes to the Sec61 channel could facilitate translocation of a secreted polypeptide and provides a valuable reference for future functional and structural studies.
References
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Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 A resolution.
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Prevention of overfitting in cryo-EM structure determination
Sjors H.W. Scheres,Shaoxia Chen +1 more
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