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
MotionCor2: Anisotropic Correction of Beam-Induced Motion for Improved Cryo-Electron Microscopy
Shawn Q. Zheng,Eugene Palovcak,Jean Paul Armache,Kliment A. Verba,Yifan Cheng,David A. Agard +5 more
TL;DR: MotionCor2 software corrects for beam-induced sample motion, improving the resolution of cryo-EM reconstructions.
Journal ArticleDOI
New tools for automated high-resolution cryo-EM structure determination in RELION-3.
Jasenko Zivanov,Takanori Nakane,Björn O. Forsberg,Dari Kimanius,Wim J. H. Hagen,Erik Lindahl,Erik Lindahl,Sjors H.W. Scheres +7 more
TL;DR: CPU-based vector acceleration has been added in addition to GPU support, which provides flexibility in use of resources and avoids memory limitations in the third major release of RELION.
Journal ArticleDOI
Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix
Dorothee Liebschner,Pavel V. Afonine,Matthew L. Baker,Gábor Bunkóczi,Vincent B. Chen,Tristan I. Croll,Bradley J. Hintze,Li-Wei Hung,Swati Jain,Airlie J. McCoy,Nigel W. Moriarty,Robert D. Oeffner,Billy K. Poon,Michael G. Prisant,Randy J. Read,Jane S. Richardson,David S. Richardson,Sammito,Oleg V. Sobolev,Duncan H. Stockwell,Thomas C. Terwilliger,Alexandre Urzhumtsev,Alexandre Urzhumtsev,Lizbeth L. Videau,Carmen J. Williams,Paul D. Adams,Paul D. Adams +26 more
TL;DR: Recent developments in the Phenix software package are described in the context of macromolecular structure determination using X-rays, neutrons and electrons.
Journal ArticleDOI
Gctf: Real-time CTF determination and correction
TL;DR: The main target of Gctf is to maximize the cross-correlation of a simulated CTF with the logarithmic amplitude spectra of observed micrographs after background subtraction to improve CTF parameters of all particles for subsequent image processing.
Journal ArticleDOI
Structure of the TRPV1 ion channel determined by electron cryo-microscopy
TL;DR: In this article, a high-resolution electron cryo-microscopy structure of the rat transient receptor potential (TRP) channel in its closed state is presented; the overall structure of this ion channel is found to share some common features with voltage-gated ion channels, although several unique, TRP-specific features are also characterized.
References
More filters
Journal ArticleDOI
The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules.
TL;DR: Because of the lack of sufficiently bright neutron sources in the foreseeable future, electron microscopy in practice provides the greatest potential for immediate progress.
Journal ArticleDOI
Flexible Fitting of Atomic Structures into Electron Microscopy Maps Using Molecular Dynamics
TL;DR: A novel method to flexibly fit atomic structures into electron microscopy (EM) maps using molecular dynamics simulations is presented, incorporating the EM data as an external potential added to the molecular dynamics force field.
Journal ArticleDOI
Nicotinic acetylcholine receptor at 4.6 A resolution: transverse tunnels in the channel wall.
TL;DR: It is suggested that the extracellular tunnels are access routes to the binding pockets for ACh, and that the cytoplasmic openings serve as filters to exclude anions and other impermeant species from the vicinity of the pore.
Journal ArticleDOI
SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs
Tanvir R. Shaikh,Haixiao Gao,William T. Baxter,Francisco J. Asturias,Nicolas Boisset,Ardean Leith,Joachim Frank,Joachim Frank,Joachim Frank +8 more
TL;DR: This protocol describes the reconstruction of biological molecules from the electron micrographs of single particles using the image-processing software SPIDER, and a method to look for homogeneous subsets when multiple known conformations of the molecule may coexist.
Journal ArticleDOI
Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles
TL;DR: It is shown that ribosome reconstructions with unprecedented resolutions may be calculated from almost two orders of magnitude fewer particles than used previously, which may expand the scope of high-resolution cryo-EM to a broad range of biological specimens.