CTFFIND4: Fast and accurate defocus estimation from electron micrographs.
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.
About: This article is published in Journal of Structural Biology.The article was published on 2015-11-01 and is currently open access. It has received 3512 citations till now. The article focuses on the topics: Contrast transfer function.
Citations
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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.
Abstract: Here, we describe the third major release of RELION. CPU-based vector acceleration has been added in addition to GPU support, which provides flexibility in use of resources and avoids memory limitations. Reference-free autopicking with Laplacian-of-Gaussian filtering and execution of jobs from python allows non-interactive processing during acquisition, including 2D-classification, de novo model generation and 3D-classification. Per-particle refinement of CTF parameters and correction of estimated beam tilt provides higher resolution reconstructions when particles are at different heights in the ice, and/or coma-free alignment has not been optimal. Ewald sphere curvature correction improves resolution for large particles. We illustrate these developments with publicly available data sets: together with a Bayesian approach to beam-induced motion correction it leads to resolution improvements of 0.2-0.7 A compared to previous RELION versions.
3,520 citations
Cites methods from "CTFFIND4: Fast and accurate defocus..."
...Other programs tackled individual steps in the single-particle workflow, like fast and robust estimation of the contrast transfer function (CTF) (Rohou and Grigorieff, 2015; Zhang, 2016), local-resolution estimation (Kucukelbir et al....
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...Because of the high tilt, Thon rings were very weak and the estimated resolution from CTFFIND4 was limited to about 10 Å....
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...In RELION, per-micrograph CTF parameters are determined through wrappers to CTFFIND (Rohou and Grigorieff, 2015) or Gctf (Zhang, 2016)....
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...We aligned and dose-weighted the 668 movies with our own implementation of the MotionCor2 algorithm, and estimated per-micrograph defocus parameters using CTFFIND4 (Rohou and Grigorieff, 2015)....
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...Currently, this requires some scripting by the user, as the wrappers to CTFFIND4 and Gctf on the RELION-3 GUI do not allow local CTF estimation....
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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.
2,919 citations
Cites methods from "CTFFIND4: Fast and accurate defocus..."
...Speed comparison was done with CTFFIND3 (Mindell and Grigorieff, 2003), CTFFIND4 (Rohou and Grigorieff, 2015), FASTDEF (Vargas et al....
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TL;DR: Eight new structures of distinct COVID-19 human neutralizing antibodies 5 in complex with the SARS-CoV-2 spike trimer or RBD are solved and rules for assigning current and future human RBD-targeting antibodies into classes, evaluating avidity effects and suggesting combinations for clinical use are provided.
Abstract: The coronavirus disease 2019 (COVID-19) pandemic presents an urgent health crisis. Human neutralizing antibodies that target the host ACE2 receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein1–5 show promise therapeutically and are being evaluated clinically6–8. Here, to identify the structural correlates of SARS-CoV-2 neutralization, we solved eight new structures of distinct COVID-19 human neutralizing antibodies5 in complex with the SARS-CoV-2 spike trimer or RBD. Structural comparisons allowed us to classify the antibodies into categories: (1) neutralizing antibodies encoded by the VH3-53 gene segment with short CDRH3 loops that block ACE2 and bind only to ‘up’ RBDs; (2) ACE2-blocking neutralizing antibodies that bind both up and ‘down’ RBDs and can contact adjacent RBDs; (3) neutralizing antibodies that bind outside the ACE2 site and recognize both up and down RBDs; and (4) previously described antibodies that do not block ACE2 and bind only to up RBDs9. Class 2 contained four neutralizing antibodies with epitopes that bridged RBDs, including a VH3-53 antibody that used a long CDRH3 with a hydrophobic tip to bridge between adjacent down RBDs, thereby locking the spike into a closed conformation. Epitope and paratope mapping revealed few interactions with host-derived N-glycans and minor contributions of antibody somatic hypermutations to epitope contacts. Affinity measurements and mapping of naturally occurring and in vitro-selected spike mutants in 3D provided insight into the potential for SARS-CoV-2 to escape from antibodies elicited during infection or delivered therapeutically. These classifications and structural analyses provide rules for assigning current and future human RBD-targeting antibodies into classes, evaluating avidity effects and suggesting combinations for clinical use, and provide insight into immune responses against SARS-CoV-2. Eight structures of human neutralizing antibodies that target the SARS-CoV-2 spike receptor-binding domain are reported and classified into four categories, suggesting combinations for clinical use.
1,169 citations
TL;DR: Two cryo–electron microscopy structures derived from a preparation of the full-length S protein, representing its prefusion and postfusion conformations, are reported, advancing the understanding of SARS-CoV-2 entry and may guide the development of vaccines and therapeutics.
Abstract: Intervention strategies are urgently needed to control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. The trimeric viral spike (S) protein catalyzes fusion between viral and target cell membranes to initiate infection. Here, we report two cryo-electron microscopy structures derived from a preparation of the full-length S protein, representing its prefusion (2.9-angstrom resolution) and postfusion (3.0-angstrom resolution) conformations, respectively. The spontaneous transition to the postfusion state is independent of target cells. The prefusion trimer has three receptor-binding domains clamped down by a segment adjacent to the fusion peptide. The postfusion structure is strategically decorated by N-linked glycans, suggesting possible protective roles against host immune responses and harsh external conditions. These findings advance our understanding of SARS-CoV-2 entry and may guide the development of vaccines and therapeutics.
903 citations
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
References
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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
"CTFFIND4: Fast and accurate defocus..." refers background or methods in this paper
...Of those, CTFFIND3 (Mindell and Grigorieff, 2003) is widely used and thought to perform well under a range of circumstances (Marabini et al., 2015)....
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...Of those, CTFFIND3 (Mindell and Grigorieff, 2003) is widely used and thought to perform well under a range of circumstances (Marabini et al....
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...We measured execution times of CTFFIND3 and CTFFIND4 with micrographs of bacteriorhodopsin, which had been used in benchmarking CTFFIND3 (Mindell and Grigorieff, 2003), as inputs....
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...They were also very low (1.75 on average) when processing bacteriorhodopsin micrographs, which have relatively large amounts of astigmatism (12–20% of Df 1; Table 2). d by crystallographic refinement of bacteriorhodopsin (Mindell and Grigorieff, 2003). in nm (Df 1;Df 2) and degrees (aast)....
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...Table 2: Comparison of defocus parameter estimates from CTFFIND3 and CTFFIND4 to those obtained by crystallographic refinement of bacteriorhodopsin (Mindell and Grigorieff, 2003)....
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TL;DR: IMAGIC's novel angular reconstitution approach allows for the rapid determination of three-dimensional structures of uncrystallized molecules to high resolution.
1,281 citations
"CTFFIND4: Fast and accurate defocus..." refers methods in this paper
...By default, input parameter values are given interactively, with a question–answer sequence inspired by that of the IMAGIC image processing package (van Heel et al., 1996), including help messages (given in response to ‘‘?”) and default answers which are updated with the user’s previous answers....
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TL;DR: Electron microscopy is a very direct method of structure determination that complements the well-established techniques of X-ray crystallography and NMR spectroscopy and is established itself as an important – still upcoming – technique for studying the structures of large biological macromolecules.
Abstract: 4. Single particles and angular reconstitution 323 4.1 Preliminary filtering and centring of data 323 4.2 Alignments using correlation functions 324 4.3 Choice of first reference images 324 4.4 Multi-reference alignment of data 325 4.5 MSA eigenvector/eigenvalue data compression 328 4.6 MSA classification 330 4.7 Euler angle determination (‘ angular reconstitution ’) 332 4.8 Sinograms and sinogram correlation functions 332 4.9 Exploiting symmetry 335 4.10 Three-dimensional reconstruction 337 4.11 Euler angles using anchor sets 339 4.12 Iterative refinements 339
627 citations
"CTFFIND4: Fast and accurate defocus..." refers methods in this paper
...To make our program more efficient, we first estimate aast separately, using an algorithm described by van Heel et al. (2000)....
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TL;DR: This primer explains the different steps and considerations involved in structure determination by single-particle cryo-EM to provide an overview for scientists wishing to understand more about this technique and the interpretation of data obtained with it, as well as a starting guide for new practitioners.
485 citations
TL;DR: The Volta phase plate has a long service life and has been used for more than 6 mo without noticeable degradation in performance, and the mechanism underlying the VPP is the same as the one responsible for the degradation over time of the performance of thin-film Zernike phase plates, but in the V PP it is used in a constructive way.
Abstract: We describe a phase plate for transmission electron microscopy taking advantage of a hitherto-unknown phenomenon, namely a beam-induced Volta potential on the surface of a continuous thin film. The Volta potential is negative, indicating that it is not caused by beam-induced electrostatic charging. The film must be heated to ∼200 °C to prevent contamination and enable the Volta potential effect. The phase shift is created “on the fly” by the central diffraction beam eliminating the need for precise phase plate alignment. Images acquired with the Volta phase plate (VPP) show higher contrast and unlike Zernike phase plate images no fringing artifacts. Following installation into the microscope, the VPP has an initial settling time of about a week after which the phase shift behavior becomes stable. The VPP has a long service life and has been used for more than 6 mo without noticeable degradation in performance. The mechanism underlying the VPP is the same as the one responsible for the degradation over time of the performance of thin-film Zernike phase plates, but in the VPP it is used in a constructive way. The exact physics and/or chemistry behind the process causing the Volta potential are not fully understood, but experimental evidence suggests that radiation-induced surface modification combined with a chemical equilibrium between the surface and residual gases in the vacuum play an important role.
439 citations
"CTFFIND4: Fast and accurate defocus..." refers background in this paper
...Volta phase plates (Danev et al., 2014) have recently become available commercially....
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