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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Book ChapterDOI
X-Ray Tomography
TL;DR: Guruswamy et al. as discussed by the authors presented a discussion of x-ray tomography using several simplifying assumptions: • We will assume parallel illumination, even though there are reconstruction algorithms [Tuy 1983, Feldkamp 1984] for cone beam tomography where the beam diverges from a point source.
Posted ContentDOI
The Shape of the Bacterial Ribosome Exit Tunnel Affects Cotranslational Protein Folding
Renuka Kudva,Pengfei Tian,Fatima Pardo Avila,Marta Carroni,Marta Carroni,Robert B. Best,Harris D. Bernstein,Gunnar von Heijne,Gunnar von Heijne +8 more
TL;DR: It is found that tunnel geometry determines where proteins of different sizes fold, which supports the idea that cotranslational folding commences once a protein domain reaches a location in the exit tunnel where there is sufficient space to house the folded structure.
Posted ContentDOI
CryoDRGN: Reconstruction of heterogeneous structures from cryo-electron micrographs using neural networks
TL;DR: This work presents cryoDRGN, an algorithm that for the first time leverages the representation power of deep neural networks to efficiently reconstruct highly heterogeneous complexes and continuous trajectories of protein motion to discover previously overlooked structural states and to visualize molecules in motion.
Journal ArticleDOI
FlhF regulates the number and configuration of periplasmic flagella in Borrelia burgdorferi.
TL;DR: Mechanistically, it is demonstrated that BB0270 localizes at the cell poles and controls the number and position of PF via regulating the flagellar protein stability and the polar localization of the MS‐ring protein FliF.
References
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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
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Journal ArticleDOI
Prevention of overfitting in cryo-EM structure determination
Sjors H.W. Scheres,Shaoxia Chen +1 more
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.