+Open data
-Basic information
Entry | Database: PDB / ID: 7kjr | |||||||||
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Title | Cryo-EM structure of SARS-CoV-2 ORF3a | |||||||||
Components |
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Keywords | TRANSPORT PROTEIN / VIRAL PROTEIN / SARS-CoV-2 / coronavirus / viroporin / membrane protein | |||||||||
Function / homology | Function and homology information host cell lysosome / induction by virus of host reticulophagy / Maturation of protein 3a / high-density lipoprotein particle receptor binding / Defective ABCA1 causes TGD / Scavenging by Class B Receptors / HDL clearance / spherical high-density lipoprotein particle / positive regulation of hydrolase activity / SARS-CoV-2 modulates autophagy ...host cell lysosome / induction by virus of host reticulophagy / Maturation of protein 3a / high-density lipoprotein particle receptor binding / Defective ABCA1 causes TGD / Scavenging by Class B Receptors / HDL clearance / spherical high-density lipoprotein particle / positive regulation of hydrolase activity / SARS-CoV-2 modulates autophagy / regulation of intestinal cholesterol absorption / negative regulation of response to cytokine stimulus / protein oxidation / vitamin transport / phosphatidylcholine-sterol O-acyltransferase activator activity / Chylomicron remodeling / positive regulation of phospholipid efflux / high-density lipoprotein particle binding / cholesterol import / Chylomicron assembly / positive regulation of cholesterol metabolic process / negative regulation of heterotypic cell-cell adhesion / high-density lipoprotein particle remodeling / blood vessel endothelial cell migration / ABC transporters in lipid homeostasis / phospholipid efflux / apolipoprotein receptor binding / high-density lipoprotein particle clearance / negative regulation of cell adhesion molecule production / negative regulation of cytokine production involved in immune response / apolipoprotein A-I receptor binding / HDL assembly / peptidyl-methionine modification / negative regulation of very-low-density lipoprotein particle remodeling / cholesterol transfer activity / reverse cholesterol transport / phosphatidylcholine biosynthetic process / high-density lipoprotein particle assembly / very-low-density lipoprotein particle / lipoprotein biosynthetic process / glucocorticoid metabolic process / positive regulation of CoA-transferase activity / phosphatidylcholine metabolic process / lipid storage / cholesterol efflux / phospholipid homeostasis / high-density lipoprotein particle / triglyceride homeostasis / regulation of Cdc42 protein signal transduction / cholesterol transport / chemorepellent activity / HDL remodeling / inorganic cation transmembrane transport / Scavenging by Class A Receptors / endothelial cell proliferation / cholesterol binding / negative regulation of interleukin-1 beta production / host cell endoplasmic reticulum / positive regulation of Rho protein signal transduction / negative chemotaxis / adrenal gland development / voltage-gated calcium channel complex / cholesterol biosynthetic process / positive regulation of cholesterol efflux / endocytic vesicle / negative regulation of tumor necrosis factor-mediated signaling pathway / Retinoid metabolism and transport / Scavenging of heme from plasma / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / voltage-gated potassium channel complex / positive regulation of phagocytosis / positive regulation of substrate adhesion-dependent cell spreading / endocytic vesicle lumen / positive regulation of stress fiber assembly / cholesterol metabolic process / heat shock protein binding / cholesterol homeostasis / molecular function activator activity / integrin-mediated signaling pathway / Post-translational protein phosphorylation / phospholipid binding / regulation of protein phosphorylation / Heme signaling / PPARA activates gene expression / negative regulation of inflammatory response / : / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / extracellular vesicle / protein complex oligomerization / Platelet degranulation / monoatomic ion channel activity / amyloid-beta binding / host cell endosome / cytoplasmic vesicle / collagen-containing extracellular matrix / blood microparticle / secretory granule lumen / Translation of Structural Proteins / Virion Assembly and Release / Induction of Cell-Cell Fusion Similarity search - Function | |||||||||
Biological species | Severe acute respiratory syndrome coronavirus 2 Homo sapiens (human) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.08 Å | |||||||||
Authors | Kern, D.M. / Hoel, C.M. / Kotecha, A. / Brohawn, S.G. | |||||||||
Funding support | United States, 2items
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Citation | Journal: Nat Struct Mol Biol / Year: 2021 Title: Cryo-EM structure of SARS-CoV-2 ORF3a in lipid nanodiscs. Authors: David M Kern / Ben Sorum / Sonali S Mali / Christopher M Hoel / Savitha Sridharan / Jonathan P Remis / Daniel B Toso / Abhay Kotecha / Diana M Bautista / Stephen G Brohawn / Abstract: SARS-CoV-2 ORF3a is a putative viral ion channel implicated in autophagy inhibition, inflammasome activation and apoptosis. 3a protein and anti-3a antibodies are found in infected patient tissues and ...SARS-CoV-2 ORF3a is a putative viral ion channel implicated in autophagy inhibition, inflammasome activation and apoptosis. 3a protein and anti-3a antibodies are found in infected patient tissues and plasma. Deletion of 3a in SARS-CoV-1 reduces viral titer and morbidity in mice, suggesting it could be an effective target for vaccines or therapeutics. Here, we present structures of SARS-CoV-2 3a determined by cryo-EM to 2.1-Å resolution. 3a adopts a new fold with a polar cavity that opens to the cytosol and membrane through separate water- and lipid-filled openings. Hydrophilic grooves along outer helices could form ion-conduction paths. Using electrophysiology and fluorescent ion imaging of 3a-reconstituted liposomes, we observe Ca-permeable, nonselective cation channel activity, identify mutations that alter ion permeability and discover polycationic inhibitors of 3a activity. 3a-like proteins are found across coronavirus lineages that infect bats and humans, suggesting that 3a-targeted approaches could treat COVID-19 and other coronavirus diseases. #1: Journal: bioRxiv / Year: 2021 Title: Cryo-EM structure of the SARS-CoV-2 3a ion channel in lipid nanodiscs. Authors: David M Kern / Ben Sorum / Sonali S Mali / Christopher M Hoel / Savitha Sridharan / Jonathan P Remis / Daniel B Toso / Abhay Kotecha / Diana M Bautista / Stephen G Brohawn / Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes the coronavirus disease 2019 (COVID-19). SARS-CoV-2 encodes three putative ion channels: E, 8a, and 3a. 3a is ...Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes the coronavirus disease 2019 (COVID-19). SARS-CoV-2 encodes three putative ion channels: E, 8a, and 3a. 3a is expressed in SARS patient tissue and anti-3a antibodies are observed in patient plasma. 3a has been implicated in viral release, inhibition of autophagy, inflammasome activation, and cell death and its deletion reduces viral titer and morbidity in mice, raising the possibility that 3a could be an effective vaccine or therapeutic target. Here, we present the first cryo-EM structures of SARS-CoV-2 3a to 2.1 Å resolution and demonstrate 3a forms an ion channel in reconstituted liposomes. The structures in lipid nanodiscs reveal 3a dimers and tetramers adopt a novel fold with a large polar cavity that spans halfway across the membrane and is accessible to the cytosol and the surrounding bilayer through separate water- and lipid-filled openings. Electrophysiology and fluorescent ion imaging experiments show 3a forms Ca-permeable non-selective cation channels. We identify point mutations that alter ion permeability and discover polycationic inhibitors of 3a channel activity. We find 3a-like proteins in multiple and lineages that infect bats and humans. These data show 3a forms a functional ion channel that may promote COVID-19 pathogenesis and suggest targeting 3a could broadly treat coronavirus diseases. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7kjr.cif.gz | 105.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7kjr.ent.gz | 75.2 KB | Display | PDB format |
PDBx/mmJSON format | 7kjr.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/kj/7kjr ftp://data.pdbj.org/pub/pdb/validation_reports/kj/7kjr | HTTPS FTP |
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-Related structure data
Related structure data | 22898MC 6xdcC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | |
EM raw data | EMPIAR-10612 (Title: High-resolution SARS-CoV-2 ORF3a dimer in an MSP1E3D1 lipid nanodisc Data size: 3.2 TB Data #1: Unaligned movies in EER format of SARS-CoV-2 3a in MSP1E3D1 lipid nanodiscs - 1379 frames [micrographs - multiframe] Data #2: Unaligned movies in EER format of SARS-CoV-2 3a in MSP1E3D1 lipid nanodiscs - 1449 frames [micrographs - multiframe]) |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | Mass: 32165.902 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2 Gene: 3a / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P0DTC3 #2: Protein | Mass: 24647.678 Da / Num. of mol.: 2 / Fragment: UNP residues 79-267 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: APOA1 / Production host: Escherichia coli (E. coli) / References: UniProt: P02647 #3: Chemical | #4: Water | ChemComp-HOH / | Has ligand of interest | N | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: SARS-CoV-2 protein 3A in lipid nanodiscsSARS-CoV-1 / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT | |||||||||||||||
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Molecular weight | Value: 0.062 MDa / Experimental value: NO | |||||||||||||||
Source (natural) | Organism: Severe acute respiratory syndrome coronavirus 2 | |||||||||||||||
Source (recombinant) | Organism: Spodoptera frugiperda (fall armyworm) | |||||||||||||||
Buffer solution | pH: 7.4 | |||||||||||||||
Buffer component |
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Specimen | Conc.: 1.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 | |||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 1 blot force 5 second wait time 3 second blot time |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy |
Image recording | Electron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) |
-Processing
Software |
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EM software | Name: PHENIX / Version: 1.18.2-3874-000 / Category: model refinement | ||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 2.08 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 91218 / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Space: REAL | ||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||
Displacement parameters | Biso mean: 42.33 Å2 | ||||||||||||||||||||||||
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