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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3.3-Å resolution cryo-EM structure of human ribonucleotide reductase with substrate and allosteric regulators bound.
Edward J. Brignole,Kuang-Lei Tsai,Johnathan Chittuluru,Haoran Li,Yimon Aye,Pawel A. Penczek,JoAnne Stubbe,Catherine L. Drennan,Francisco J. Asturias +8 more
TL;DR: A 3.3-Å resolution structure by cryo-electron microscopy of a dATP-inhibited state of human RNR supports a model for RNR inhibition in which β2 is excluded from binding in a radical transfer competent position when α exists as a stable hexamer.
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The potential of cryo-electron microscopy for structure-based drug design
TL;DR: The potential of cryo-EM for drug discovery is reviewed with reference to protein ligand complex structures determined using this technique, with a focus on proteins as small as 64 kDa.
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Structural insight into microtubule stabilization and kinesin inhibition by Tau family MAPs.
Hideki Shigematsu,Tsuyoshi Imasaki,Tsuyoshi Imasaki,Chihiro Doki,Takuya Sumi,Mari Aoki,Tomomi Uchikubo-Kamo,Ayako Sakamoto,Kiyotaka Tokuraku,Mikako Shirouzu,Ryo Nitta,Ryo Nitta +11 more
TL;DR: Cryo-EM studies reveal new insight into the structural basis of microtubule stabilization and inhibition of kinesin motility by the Tau family MAPs.
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2dx_automator: Implementation of a semiautomatic high-throughput high-resolution cryo-electron crystallography pipeline
Sebastian Scherer,Julia Kowal,Mohamed Chami,Venkata P. Dandey,Marcel Arheit,Philippe Ringler,Henning Stahlberg +6 more
TL;DR: The optimal use of DEDs for cryo-electron crystallography is discussed, a new automatic image processing pipeline is introduced, and the vast improvement in the resolution achieved by the use of both together is demonstrated.
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Structural basis for ArfA–RF2-mediated translation termination on mRNAs lacking stop codons
Paul Huter,Claudia Müller,Bertrand Beckert,Bertrand Beckert,Stefan Arenz,Otto Berninghausen,Roland Beckmann,Daniel N. Wilson,Daniel N. Wilson +8 more
TL;DR: A cryo-electron microscopy reconstruction of the Escherichia coli 70S ribosome stalled on a truncated mRNA in the presence of ArfA and RF2 reveals not only howArfA recruits RF2 to the ribosomes but also how it promotes an active conformation ofRF2 to enable translation termination in the absence of a stop codon.
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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