Real-time cryo-electron microscopy data preprocessing with Warp

Tegunov, Dimitry; Cramer, Patrick

doi:10.1038/s41592-019-0580-y

Cited by 1,080 publications

(934 citation statements)

References 63 publications

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“…Cryo-EM data processing Movie frame alignment, estimation of the microscope contrast-transfer function parameters, particle picking and extraction were carried out using Warp (Tegunov and Cramer, 2019). Particle images were extracted with a box size of 800 binned to 400 yielding a pixel size of 1.05 Å .…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

Walls

Park

Tortorici

et al. 2020

Cell

8,011

258

8,384

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Graphical AbstractHighlights d SARS-CoV-2 uses ACE2 to enter target cells d SARS-CoV-2 and SARS-CoV bind with similar affinities to ACE2 d Structures of SARS-CoV-2 spike glycoprotein in two conformations d SARS-CoV polyclonal antibodies inhibit SARS-CoV-2 spikemediated entry into cellsIn Brief SARS-CoV-2, a newly emerged pathogen spreading worldwide, binds with high affinity to human ACE2 and uses it as an entry receptor to invade target cells.Cryo-EM structures of the SARS-CoV-2 spike glycoprotein in two distinct conformations, along with inhibition of spike-mediated entry by SARS-CoV polyclonal antibodies, provide a blueprint for the design of vaccines and therapeutics. SUMMARYThe emergence of SARS-CoV-2 has resulted in >90,000 infections and >3,000 deaths. Coronavirus spike (S) glycoproteins promote entry into cells and are the main target of antibodies. We show that SARS-CoV-2 S uses ACE2 to enter cells and that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, correlating with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S 1 /S 2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and SARS-related CoVs. We determined cryo-EM structures of the SARS-CoV-2 S ectodomain trimer, providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal antibodies potently inhibited SARS-CoV-2 S mediated entry into cells, indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.

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Section: Protein Expression and Purificationmentioning

confidence: 99%

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

Walls

Park

Tortorici

et al. 2020

Cell

8,011

258

8,384

View full text Add to dashboard Cite

show abstract

“…Motion correction, CTF-estimation and non-templated particle picking were performed in Warp 158 (32). Extracted particles were imported into cryoSPARC v2.12.4 (15) for 2D classification, 3D 159 classification and non-uniform 3D refinement.…”

Section: Cryo-em Data Processing 157mentioning

confidence: 99%

Cryo-EM Structure of the 2019-nCoV Spike in the Prefusion Conformation

Wrapp

Wang

Corbett

et al. 2020

Preprint

1,351

1,910

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1The outbreak of a novel betacoronavirus (2019-nCov) represents a pandemic threat that has been 2 declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a 3 key target for urgently needed vaccines, therapeutic antibodies, and diagnostics. To facilitate 4 medical countermeasure (MCM) development we determined a 3.5 Å-resolution cryo-EM 5 structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the 6 trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible 7 conformation. We also show biophysical and structural evidence that the 2019-nCoV S binds 8 ACE2 with higher affinity than SARS-CoV S. Additionally we tested several published SARS-9CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to 10 nCoV-2019 S, suggesting antibody cross-reactivity may be limited between the two virus RBDs. 11The atomic-resolution structure of 2019-nCoV S should enable rapid development and evaluation

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“…Movies were pre-processed on the fly using Warp 1.05 27 . The model parameters for motion correction and CTF estimation were set to 5×5×40 and 5×5×1, respectively.…”

Section: Electron Microscopymentioning

confidence: 99%

Structural basis of RNA polymerase III transcription repression by Maf1

Vorländer¹,

Baudin²,

Moir³

et al. 2019

Preprint

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Maf1 is a highly conserved central regulator of transcription by RNA polymerase III (Pol III), and Maf1 activity influences a wide range of phenotypes from metabolic efficiency to lifespan. Here, we present a 3.3 Å cryo-EM structure of yeast Maf1 bound to Pol III, which establishes how Maf1 achieves transcription repression. In the Maf1-bound state, Pol III elements that are involved in transcription initiation are sequestered, and the active site is sealed off due to ordering of the mobile C34 winged helix 2 domain. Specifically, the Maf1 binding site overlaps with the binding site of the Pol III transcription factor TFIIIB and DNA in the pre-initiation complex, rationalizing that binding of Maf1 and TFIIIB to Pol III are mutually exclusive. We validate our structure using variants of Maf1 with impaired transcription-inhibition activity. These results reveal the exact mechanism of Pol III inhibition by Maf1, and rationalize previous biochemical data.

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Real-time cryo-electron microscopy data preprocessing with Warp

Cited by 1,080 publications

References 63 publications

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

Cryo-EM Structure of the 2019-nCoV Spike in the Prefusion Conformation

Structural basis of RNA polymerase III transcription repression by Maf1

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