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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Determining the bacterial cell biology of Planctomycetes
Christian Boedeker,Margarete Schüler,Greta Reintjes,Olga Jeske,Muriel C. F. van Teeseling,Muriel C. F. van Teeseling,Mareike Jogler,Patrick Rast,Daniela Borchert,Damien P. Devos,Martin Kucklick,Miroslava Schaffer,Roberto Kolter,Laura van Niftrik,Susanne Engelmann,Rudolf Amann,Manfred Rohde,Harald Engelhardt,Christian Jogler,Christian Jogler +19 more
TL;DR: Methods for expression of fluorescent proteins and for gene deletion in a model planctomycete, Planctopirus limnophila, to analyse its cell organization in detail are developed and experimental evidence is provided that is inconsistent with endocytosis-like macromolecule uptake.
Journal ArticleDOI
Lateral opening in the intact β-barrel assembly machinery captured by cryo-EM.
Matthew G. Iadanza,Anna J. Higgins,Bob Schiffrin,Antonio N. Calabrese,David J. Brockwell,Alison E. Ashcroft,Sheena E. Radford,Neil A. Ranson +7 more
TL;DR: It is shown that for the outer membrane protein OmpT, efficient folding in vitro requires lateral gating in BAM, and conformational changes throughout the complex and interactions between BamA, B, D and E, and the detergent micelle that suggest communication between BAM and the lipid bilayer are described.
Journal ArticleDOI
Human SEIPIN Binds Anionic Phospholipids.
Renhong Yan,Hongwu Qian,Ivan Lukmantara,Mingming Gao,Ximing Du,Nieng Yan,Nieng Yan,Hongyuan Yang +7 more
TL;DR: The results suggest that SEIPIN forms a scaffold that helps maintain phospholipid homeostasis and surface tension of the ER.
Journal ArticleDOI
Localization events-based sample drift correction for localization microscopy with redundant cross-correlation algorithm.
TL;DR: This paper presents a localization events-based drift correction method using a redundant cross-correlation algorithm originally developed to correct beam-induced motion in cryo-electron microscopy to improve the effective resolution when imaging Golgi apparatus in mammalian cells.
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Cryo-EM single particle analysis with the Volta phase plate.
TL;DR: A method for in-focus data acquisition with a phase plate that enables near-atomic resolution single particle reconstructions and could enable single particle analysis of challenging samples in terms of small size, heterogeneity and flexibility that are difficult to solve by the conventional defocus approach.
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.
Journal ArticleDOI
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.
Journal ArticleDOI
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.
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.