Automated electron microscope tomography using robust prediction of specimen movements.
TL;DR: A new method was developed to acquire images automatically at a series of specimen tilts, as required for tomographic reconstruction, using changes in specimen position at previous tilt angles to predict the position at the current tilt angle.
About: This article is published in Journal of Structural Biology.The article was published on 2005-10-01. It has received 3995 citations till now. The article focuses on the topics: Tomographic reconstruction & Tilt (optics).
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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
TL;DR: Most convalescent plasma samples obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity, and rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.
Abstract: During the COVID-19 pandemic, SARS-CoV-2 infected millions of people and claimed hundreds of thousands of lives Virus entry into cells depends on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S) Although there is no vaccine, it is likely that antibodies will be essential for protection However, little is known about the human antibody response to SARS-CoV-21-5 Here we report on 149 COVID-19 convalescent individuals Plasmas collected an average of 39 days after the onset of symptoms had variable half-maximal pseudovirus neutralizing titres: less than 1:50 in 33% and below 1:1,000 in 79%, while only 1% showed titres above 1:5,000 Antibody sequencing revealed expanded clones of RBD-specific memory B cells expressing closely related antibodies in different individuals Despite low plasma titres, antibodies to three distinct epitopes on RBD neutralized at half-maximal inhibitory concentrations (IC50 values) as low as single digit nanograms per millitre Thus, most convalescent plasmas obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective
1,675 citations
TL;DR: The results show that atheroprotective stimuli induce communication between endothelial cells and SMCs through an miRNA- and extracellular-vesicle-mediated mechanism and that this may comprise a promising strategy to combat atherosclerosis.
Abstract: The shear-responsive transcription factor Kruppel-like factor 2 (KLF2) is a critical regulator of endothelial gene expression patterns induced by atheroprotective flow. As microRNAs (miRNAs) post-transcriptionally control gene expression in many pathogenic and physiological processes, we investigated the regulation of miRNAs by KLF2 in endothelial cells. KLF2 binds to the promoter and induces a significant upregulation of the miR-143/145 cluster. Interestingly, miR-143/145 has been shown to control smooth muscle cell (SMC) phenotypes; therefore, we investigated the possibility of transport of these miRNAs between endothelial cells and SMCs. Indeed, extracellular vesicles secreted by KLF2-transduced or shear-stress-stimulated HUVECs are enriched in miR-143/145 and control target gene expression in co-cultured SMCs. Extracellular vesicles derived from KLF2-expressing endothelial cells also reduced atherosclerotic lesion formation in the aorta of ApoE(-/-) mice. Combined, our results show that atheroprotective stimuli induce communication between endothelial cells and SMCs through an miRNA- and extracellular-vesicle-mediated mechanism and that this may comprise a promising strategy to combat atherosclerosis.
1,182 citations
Cites methods from "Automated electron microscope tomog..."
...Electron tomograms were recorded using the SerialEM softwar...
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TL;DR: The structure of the COVID-19 virus polymerase essential for viral replication provides a basis for the design of new antiviral drugs that target viral RdRp, also named nsp12, and it appears to be a primary target for the antiviral drug remdesivir.
Abstract: A novel coronavirus (COVID-19 virus) outbreak has caused a global pandemic resulting in tens of thousands of infections and thousands of deaths worldwide. The RNA-dependent RNA polymerase (RdRp, also named nsp12) is the central component of coronaviral replication/transcription machinery and appears to be a primary target for the antiviral drug, remdesivir. We report the cryo-EM structure of COVID-19 virus full-length nsp12 in complex with cofactors nsp7 and nsp8 at 2.9-A resolution. In addition to the conserved architecture of the polymerase core of the viral polymerase family, nsp12 possesses a newly identified β-hairpin domain at its N terminus. A comparative analysis model shows how remdesivir binds to this polymerase. The structure provides a basis for the design of new antiviral therapeutics targeting viral RdRp.
1,180 citations
Cites methods from "Automated electron microscope tomog..."
...All images were automatically recorded using SerialEM (18)....
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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
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
"Automated electron microscope tomog..." refers methods in this paper
...This angle is obtained from the fiducial marker alignment (Lawrence, 1992) of a typical tilt series using the IMOD software (Kremer et al., 1996; Mastronarde, 1997)....
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TL;DR: Analysis by Fourier sector correlation indicated that the variable tilt increment improved the reconstruction in some respects but degraded it in others, and a varying tilt increment thus does not give an unqualified improvement, at least when using back-projection algorithms for the reconstruction.
1,029 citations
"Automated electron microscope tomog..." refers methods in this paper
...This angle is obtained from the fiducial marker alignment (Lawrence, 1992) of a typical tilt series using the IMOD software (Kremer et al., 1996; Mastronarde, 1997)....
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TL;DR: TOM software toolbox integrates established algorithms and new concepts tailored to the special needs of low dose ET, which provides a user-friendly unified platform for all processing steps: acquisition, alignment, reconstruction, and analysis.
440 citations
"Automated electron microscope tomog..." refers methods in this paper
...For tomography of beam-sensitive specimens such as frozen-hydrated material, the standard approach has been to take focus and tracking images at a location displaced from the area of interest along the tilt axis (Dierksen et al., 1993; Nickell et al., 2005; Rath et al., 1997)....
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TL;DR: To be of maximal value, structural studies should provide isotropic, 3D information about well-preserved samples at the highest possible resolution about many kinds of cells and organelles.
405 citations
"Automated electron microscope tomog..." refers methods in this paper
...Electron tomography has become increasingly important as a technique for examining cellular structures and macromolecules in three dimensions (Baumeister, 2002; Leapman, 2004; McIntosh et al., 2005; Steven and Aebi, 2003)....
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TL;DR: After a general introduction to three-dimensional electron microscopy and particularly to electron tomography (ET), the perspectives of applying ET to native (frozen-hydrated) cellular structures are discussed and the question of whether it is a realistic long-term goal to visualize or identify macromolecules in cells frozen in toto or in frozen sections of cells is addressed.
403 citations
"Automated electron microscope tomog..." refers background in this paper
...Energy filtering has been found to be valuable for imaging both thick frozen-hydrated specimens (Grimm et al., 1997; Koster et al., 1997) and stained plastic sections (Bouwer et al., 2004; Han et al., 1996)....
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...Energy filtering has been found to be valuable for imaging both thick frozen-hydrated specimens (Grimm et al., 1997; Koster et al., 1997) and stained plastic sections (Bouwer et al....
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