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
Reads0
Chats0
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
More filters
Journal ArticleDOI
Golgi apparatus analyzed by cryo-electron microscopy
TL;DR: Even though the overall morphology of vitrified Golgi stacks is comparable to well-prepared and resin-embedded samples, previously unknown structural details can be observed solely based on their native density and any further improvement of sample preparation would gain novel insights.
Posted ContentDOI
Vimentin Intermediate Filaments and Filamentous Actin Form Unexpected Interpenetrating Networks That Redefine the Cell Cortex
Huayin Wu,Yinan Shen,Suganya Sivagurunathan,Miriam Weber,Stephen A. Adam,Jennifer Hyunjong Shin,Jeffrey J. Fredberg,Ohad Medalia,Robert D. Goldman,David A. Weitz +9 more
TL;DR: In this paper, it was shown that VIFs and F-actin form an interpenetrating network within the cell cortex and interact synergistically at multiple length scales.
Journal ArticleDOI
Cryo-EM studies of the structure and dynamics of vacuolar-type ATPases
TL;DR: This Review will discuss what has been learned recently about rotary ATPase structure and function, with a particular focus on the vacuolar-type ATPases.
Journal ArticleDOI
Structural impact of K63 ubiquitin on yeast translocating ribosomes under oxidative stress
Ye Zhou,Panagiotis L. Kastritis,Shannon E. Dougherty,Jonathan Bouvette,Allen L. Hsu,Laura Burbaum,Shyamal Mosalaganti,Stefan Pfeffer,Wim J. H. Hagen,Friedrich Förster,Friedrich Förster,Mario J. Borgnia,Christine Vogel,Martin Beck,Alberto Bartesaghi,Gustavo M. Silva +15 more
TL;DR: An examination of the structure of K63 ubiquitinated ribosome reveals that this modification structurally destabilizes proteins involved in the binding of translation factors and is required to trap ribosomes at the pretranslocation stage of translation elongation, providing evidence for a new redox regulatory mechanism of translation.
Journal ArticleDOI
Functional reconstitution of the type IVa pilus assembly system from enterohaemorrhagic Escherichia coli.
TL;DR: Bacterial two‐hybrid analysis identified several interactions of PpdD with T4aP assembly proteins, and with components of the T2SS that allow for heterologous fibre assembly.
References
More filters
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.