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
Posted ContentDOI
Simultaneous Determination of Protein Structure and Dynamics Using Cryo-Electron Microscopy
TL;DR: An integrative modelling approach is reported to simultaneously determine structure and dynamics from cryo-electron microscopy density maps and provide insights into the mechanisms by which the integral membrane receptor STRA6 interacts with retinol binding protein and translocatesretinol across the membrane.
Journal ArticleDOI
Evaluation of super-resolution performance of the K2 electron-counting camera using 2D crystals of aquaporin-0.
Po-Lin Chiu,Xueming Li,Zongli Li,Brian C Beckett,Axel F. Brilot,Nikolaus Grigorieff,David A. Agard,Yifan Cheng,Thomas Walz,Thomas Walz +9 more
TL;DR: Well-characterized two-dimensional crystals of the membrane protein aquaporin-0 are used to characterize the performance of the camera below and beyond the physical Nyquist limit and to measure the influence of electron dose rate on image amplitudes and phases.
Journal ArticleDOI
Transmission electron microscopy of cellulose. Part 2: technical and practical aspects
Yu Ogawa,Jean-Luc Putaux +1 more
TL;DR: In this article, practical aspects of sample preparation, contrast enhancing protocols as well as specific imaging and diffraction techniques are described to facilitate the morphological and structural characterization of cellulose by TEM in imaging and electron diffraction modes.
Journal ArticleDOI
Visualizing the structure and motion of the long noncoding RNA HOTAIR.
Rachel Spokoini-Stern,Dimitar R. Stamov,Hadass R. Jessel,Lior Aharoni,Heiko Haschke,Jonathan Giron,Ron Unger,Eran Segal,Almogit Abu-Horowitz,Ido Bachelet +9 more
TL;DR: This report provides a biologically-plausible description of the anatomy and intrinsic properties of HOTAIR, and presents a framework for studying the structural biology of lncRNAs.
Journal ArticleDOI
Development of a Novel Virus-Like Particle Vaccine Platform That Mimics the Immature Form of Alphavirus.
Akane Urakami,Atsuko Sakurai,Momoko Ishikawa,Moh Lan Yap,Yevel Flores-Garcia,Yasunari Haseda,Taiki Aoshi,Fidel Zavala,Michael G. Rossmann,Sachiko Kuno,Ryuji Ueno,Wataru Akahata +11 more
TL;DR: This work developed a novel VLP-based vaccine platform utilizing VLPs from the chikungunya virus and used the circumsporozoite protein (CSP) of the Plasmodium falciparum malaria parasite as an antigen and demonstrated that it elicits strong immune responses against CSP in animals.
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.