Efficient 3D-CTF correction for cryo-electron tomography using NovaCTF improves subtomogram averaging resolution to 3.4Å.
TL;DR: A user-friendly, computationally-efficient 3D-CTF correction tool, NovaCTF, that is compatible with standard tomogram reconstruction workflows in IMOD is presented and it is found that high-resolution structures can be obtained with much smaller subtomogram averaging datasets than are required using 2D- CTF.
About: This article is published in Journal of Structural Biology.The article was published on 2017-09-01 and is currently open access. It has received 198 citations till now.
Citations
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TL;DR: Cryo-electron microscopy and tomography is applied to image intact SARS-CoV-2 virions, determining the high-resolution structure, conformational flexibility and distribution of S trimers in situ on the virion surface and providing a basis from which to understand interactions between S and neutralizing antibodies during infection or vaccination.
Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude1. Heavily glycosylated S trimers bind to the angiotensin-converting enzyme 2 receptor and mediate entry of virions into target cells2–6. S exhibits extensive conformational flexibility: it modulates exposure of its receptor-binding site and subsequently undergoes complete structural rearrangement to drive fusion of viral and cellular membranes2,7,8. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy2,7,9–12, but the structure and distribution of S on the virion surface remain unknown. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions and determine the high-resolution structure, conformational flexibility and distribution of S trimers in situ on the virion surface. These results reveal the conformations of S on the virion, and provide a basis from which to understand interactions between S and neutralizing antibodies during infection or vaccination. Cryo-electron microscopy and tomography studies reveal the structures, conformations and distributions of spike protein trimers on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions and provide a basis for understanding the interactions of the spike protein with neutralizing antibodies.
808 citations
TL;DR: Warp is described, a software that automates all preprocessing steps of cryo-EM data acquisition and enables real-time evaluation, and includes deep-learning-based models for accurate particle picking and image denoising.
Abstract: The acquisition of cryo-electron microscopy (cryo-EM) data from biological specimens must be tightly coupled to data preprocessing to ensure the best data quality and microscope usage. Here we describe Warp, a software that automates all preprocessing steps of cryo-EM data acquisition and enables real-time evaluation. Warp corrects micrographs for global and local motion, estimates the local defocus and monitors key parameters for each recorded micrograph or tomographic tilt series in real time. The software further includes deep-learning-based models for accurate particle picking and image denoising. The output from Warp can be fed into established programs for particle classification and 3D-map refinement. Our benchmarks show improvement in the nominal resolution, which went from 3.9 A to 3.2 A, of a published cryo-EM data set for influenza virus hemagglutinin. Warp is easy to install from http://github.com/cramerlab/warp
and computationally inexpensive, and has an intuitive, streamlined user interface. The user-friendly software tool Warp enables automated, on-the-fly preprocessing of cryo-EM data, including motion correction, defocus estimation, particle picking and image denoising.
655 citations
TL;DR: Overall, these characterizations have revealed the architecture of the SARS-CoV-2 virus in exceptional detail, and shed lights on how the virus packs its ∼30 kb long single-segmented RNA in the ∼80 nm diameter lumen.
646 citations
TL;DR: The stalk domain of S contains three hinges, giving the head unexpected orientational freedom and it is proposed that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat.
Abstract: The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the primary focus for vaccine development. In this study, we combined cryo-electron tomography, subtomogram averaging, and molecular dynamics simulations to structurally analyze S in situ. Compared with the recombinant S, the viral S was more heavily glycosylated and occurred mostly in the closed prefusion conformation. We show that the stalk domain of S contains three hinges, giving the head unexpected orientational freedom. We propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and potentially to the development of safe vaccines.
461 citations
TL;DR: Overall, these characterizations have revealed the architecture of the SARS-CoV-2 virus in unprecedented detail, and shed lights on how the virus packs its ∼30 kb long single-segmented RNA in the ∼80 nm diameter lumen.
Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped virus responsible for the COVID-19 pandemic. Despite recent advances in the structural elucidation of SARS-CoV-2 proteins and the complexes of the spike (S) proteins with the cellular receptor ACE2 or neutralizing antibodies, detailed architecture of the intact virus remains to be unveiled. Here we report the molecular assembly of the authentic SARS-CoV-2 virus using cryo-electron tomography (cryo-ET) and subtomogram averaging (STA). Native structures of the S proteins in both pre- and postfusion conformations were determined to average resolutions of 9-11 A. Compositions of the N-linked glycans from the native spikes were analyzed by mass spectrometry, which revealed highly similar overall processing states of the native glycans to that of the recombinant glycoprotein glycans. The in situ architecture of the ribonucleoproteins (RNP) and its higher-order assemblies were revealed. These characterizations have revealed the architecture of the SARS-CoV-2 virus to an unprecedented resolution, and shed lights on how the virus packs its ~30 Kb long single-segmented RNA in the ~80 nm diameter lumen. Overall, the results unveiled the molecular architecture and assembly of the SARS-CoV-2 in native context.
307 citations
References
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TL;DR: IMOD is useful for studying and modeling data from tomographic, serial section, and optical section reconstructions and allows image data to be visualized by several different methods.
4,830 citations
TL;DR: Modifications to the CTFFIND algorithm are described which make it significantly faster and more suitable for use with images collected using modern technologies such as dose fractionation and phase plates.
3,512 citations
TL;DR: This implementation of the Algebraic Reconstruction Technique appears to have a computational advantage over the more traditional implementation of ART and potential applications include image reconstruction in conjunction with ray tracing for ultrasound and microwave tomography.
1,539 citations
"Efficient 3D-CTF correction for cry..." refers background or methods in this paper
...Recently Kunz and Frangakis (2017) proposed a GPU implementation of 3D-CTF correction related to the Jensen and Kornberg (2000) approach in combination with two different reconstruction techniques: Weighted Back Projection (WBP) (Bracewell and Riddle, 1967; Crowther et al., 1970; Gilbert, July 1972) and Simultaneous Algebraic Reconstruction Technique (SART) (Andersen and Kak, 1984)....
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...…related to the Jensen and Kornberg (2000) approach in combination with two different reconstruction techniques: Weighted Back Projection (WBP) (Bracewell and Riddle, 1967; Crowther et al., 1970; Gilbert, July 1972) and Simultaneous Algebraic Reconstruction Technique (SART) (Andersen and Kak, 1984)....
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..., 1970; Gilbert, July 1972) and Simultaneous Algebraic Reconstruction Technique (SART) (Andersen and Kak, 1984)....
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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.
1,480 citations
Book•
15 Aug 1949
TL;DR: Extrapolation interpolation and smoothing of stationary, stationary tones interference cancellation using adaptive and stationary time series financial definition of stationary.
1,332 citations
"Efficient 3D-CTF correction for cry..." refers methods in this paper
...This can be achieved by phase-flipping, where the sign is simply inverted; by multiplication with the CTF; or by Wiener filtering (Wiener, 1949), where CTF is divided by a frequency-dependent weighting function....
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