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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Structural inhibition of dynamin-mediated membrane fission by endophilin.
Annika Hohendahl,Nathaniel Talledge,Valentina Galli,Peter S. Shen,Frédéric Humbert,Pietro De Camilli,Adam Frost,Aurélien Roux +7 more
TL;DR: It is shown that dynamin-mediated membrane fission is potently inhibited in vitro when an excess of endophilin co-assembles with dynamin around membrane tubules and intercalates between turns of the dynamin helix and impairs fission by preventing trans interactions between dynamin rungs that are thought to play critical roles in membrane constriction.
Posted Content
Alignment of cryo-EM movies of individual particles by optimization of image translations
TL;DR: An algorithm is described that allows for individual <1 MDa particle images to be aligned without frame averaging or linear trajectories, and can be used to improve 3-D maps from single particle cryo-EM.
Journal ArticleDOI
Benchmarking tomographic acquisition schemes for high-resolution structural biology
Beata Turoňová,Wim J. H. Hagen,Martin Obr,Martin Obr,Shyamal Mosalaganti,J. Wouter Beugelink,Christian E. Zimmerli,Hans-Georg Kräusslich,Martin Beck +8 more
TL;DR: The authors systematically benchmark cryo-electron tomography acquisition schemes to optimize the attainable resolution for subtomogram averaging, and find that dose-symmetric acquisition with even angular sampling provides a better outcome than most currently used acquisition schemes.
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Identifying and Visualizing Macromolecular Flexibility in Structural Biology
TL;DR: How current structural biology methods can contribute useful data to accurately visualize flexibility in macromolecular structures and understand its important roles in regulation of biological processes is reviewed.
Journal ArticleDOI
Hybrid methods for macromolecular structure determination: experiment with expectations.
TL;DR: This review describes recent approaches for building atomic models with a focus on X-ray diffraction and single-particle cryo-electron microscopy (cryo-EM) data, and suggests that using raw data for the modeling ensures that all information obtained by the experiment can be fully exploited.
References
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Journal ArticleDOI
UCSF Chimera--a visualization system for exploratory research and analysis.
Eric F. Pettersen,Thomas D. Goddard,Conrad C. Huang,Gregory S. Couch,Daniel M. Greenblatt,Elaine C. Meng,Thomas E. Ferrin +6 more
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.
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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.