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- PDB-7kjr: Cryo-EM structure of SARS-CoV-2 ORF3a -

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Basic information

Entry
Database: PDB / ID: 7kjr
TitleCryo-EM structure of SARS-CoV-2 ORF3a
Components
  • Apolipoprotein A-I
  • ORF3a protein
KeywordsTRANSPORT PROTEIN / VIRAL PROTEIN / SARS-CoV-2 / coronavirus / viroporin / membrane protein
Function / homology
Function and homology information


host cell lysosome / symbiont-mediated activation of host reticulophagy / Maturation of protein 3a / Defective ABCA1 causes TGD / high-density lipoprotein particle receptor binding / HDL clearance / peptidyl-methionine modification / spherical high-density lipoprotein particle / Scavenging by Class B Receptors / negative regulation of response to cytokine stimulus ...host cell lysosome / symbiont-mediated activation of host reticulophagy / Maturation of protein 3a / Defective ABCA1 causes TGD / high-density lipoprotein particle receptor binding / HDL clearance / peptidyl-methionine modification / spherical high-density lipoprotein particle / Scavenging by Class B Receptors / negative regulation of response to cytokine stimulus / protein oxidation / regulation of intestinal cholesterol absorption / vitamin transport / blood vessel endothelial cell migration / cholesterol import / SARS-CoV-2 modulates autophagy / high-density lipoprotein particle binding / negative regulation of heterotypic cell-cell adhesion / ABC transporters in lipid homeostasis / apolipoprotein A-I receptor binding / apolipoprotein receptor binding / negative regulation of cell adhesion molecule production / negative regulation of cytokine production involved in immune response / negative regulation of very-low-density lipoprotein particle remodeling / HDL assembly / phosphatidylcholine biosynthetic process / glucocorticoid metabolic process / acylglycerol homeostasis / phosphatidylcholine-sterol O-acyltransferase activator activity / positive regulation of phospholipid efflux / Chylomicron remodeling / cellular response to lipoprotein particle stimulus / Chylomicron assembly / high-density lipoprotein particle clearance / chylomicron / phospholipid efflux / high-density lipoprotein particle remodeling / lipid storage / positive regulation of cholesterol metabolic process / reverse cholesterol transport / phospholipid homeostasis / chemorepellent activity / high-density lipoprotein particle assembly / low-density lipoprotein particle / lipoprotein biosynthetic process / cholesterol transfer activity / cholesterol transport / high-density lipoprotein particle / very-low-density lipoprotein particle / endothelial cell proliferation / regulation of Cdc42 protein signal transduction / inorganic cation transmembrane transport / HDL remodeling / cholesterol efflux / triglyceride homeostasis / voltage-gated calcium channel complex / Scavenging by Class A Receptors / negative regulation of interleukin-1 beta production / adrenal gland development / negative chemotaxis / host cell endoplasmic reticulum / cholesterol binding / cholesterol biosynthetic process / positive regulation of Rho protein signal transduction / amyloid-beta formation / monoatomic ion channel activity / positive regulation of cholesterol efflux / endocytic vesicle / negative regulation of tumor necrosis factor-mediated signaling pathway / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / Scavenging of heme from plasma / Retinoid metabolism and transport / positive regulation of stress fiber assembly / positive regulation of phagocytosis / heat shock protein binding / voltage-gated potassium channel complex / endocytic vesicle lumen / positive regulation of substrate adhesion-dependent cell spreading / cholesterol metabolic process / cholesterol homeostasis / molecular function activator activity / integrin-mediated signaling pathway / Post-translational protein phosphorylation / Heme signaling / PPARA activates gene expression / phospholipid binding / negative regulation of inflammatory response / cytoplasmic side of plasma membrane / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / Platelet degranulation / extracellular vesicle / amyloid-beta binding / host cell endosome / cytoplasmic vesicle / : / secretory granule lumen / blood microparticle / Translation of Structural Proteins / Virion Assembly and Release / Induction of Cell-Cell Fusion
Similarity search - Function
Protein 3a, betacoronavirus / 3a-like viroporin, transmembrane domain, alpha/betacoronavirus / 3a-like viroporin, cytosolic domain, alpha/betacoronavirus / Betacoronavirus viroporin / Coronavirus (CoV) 3a-like viroporin trans-membrane (TM) domain profile. / Coronavirus (CoV) 3a-like viroporin cytosolic (CD) domain profile. / Apolipoprotein A/E / : / Apolipoprotein A1/A4/E domain
Similarity search - Domain/homology
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / Apolipoprotein A-I / ORF3a protein
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.08 Å
AuthorsKern, D.M. / Hoel, C.M. / Kotecha, A. / Brohawn, S.G.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM123496 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM128263 United States
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
DepositionOct 26, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 18, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 27, 2021Group: Structure summary / Category: entity / entity_name_com / Item: _entity.pdbx_description / _entity_name_com.name
Revision 1.2Aug 18, 2021Group: Database references / Category: citation / citation_author / database_2
Item: _citation.year / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 2.0Aug 31, 2022Group: Advisory / Atomic model ...Advisory / Atomic model / Database references / Derived calculations / Non-polymer description / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / citation / citation_author / entity / pdbx_entity_nonpoly / pdbx_unobs_or_zero_occ_atoms
Item: _atom_site.Cartn_x / _atom_site.Cartn_y ..._atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_atom_id / _atom_site.label_atom_id / _atom_site.type_symbol / _chem_comp.formula / _chem_comp.formula_weight / _chem_comp.mon_nstd_flag / _chem_comp.name / _entity.formula_weight / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _pdbx_unobs_or_zero_occ_atoms.auth_atom_id / _pdbx_unobs_or_zero_occ_atoms.label_atom_id
Revision 2.1May 29, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / citation / Item: _citation.journal_id_ISSN

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Structure visualization

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Assembly

Deposited unit
A: ORF3a protein
B: ORF3a protein
C: Apolipoprotein A-I
D: Apolipoprotein A-I
hetero molecules


Theoretical massNumber of molelcules
Total (without water)115,1156
Polymers113,6274
Non-polymers1,4882
Water2,198122
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9370 Å2
ΔGint-82 kcal/mol
Surface area22010 Å2

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Components

#1: Protein ORF3a protein / Accessory protein 3a / Protein U274 / Protein X1 / ORF3a / Protein 3a


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 Apolipoprotein A-I / ApoA-I / Apolipoprotein A1 / MSPE3D1


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 ChemComp-PEE / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE


Mass: 744.034 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C41H78NO8P / Comment: DOPE, phospholipid*YM
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 122 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: SARS-CoV-2 protein 3A in lipid nanodiscs / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.062 MDa / Experimental value: NO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMPotassium ChlorideKCl1
SpecimenConc.: 1.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: 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

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
EM softwareName: PHENIX / Version: 1.18.2-3874-000 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.08 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 91218 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingSpace: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 42.33 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00533818
ELECTRON MICROSCOPYf_angle_d0.70835186
ELECTRON MICROSCOPYf_chiral_restr0.0464576
ELECTRON MICROSCOPYf_plane_restr0.0049626
ELECTRON MICROSCOPYf_dihedral_angle_d8.6355502

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