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- EMDB-22898: Cryo-EM structure of SARS-CoV-2 ORF3a -

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Basic information

Entry
Database: EMDB / ID: EMD-22898
TitleCryo-EM structure of SARS-CoV-2 ORF3a
Map dataSharpened and locally filtered map
Sample
  • Complex: SARS-CoV-2 protein 3A in lipid nanodiscsSARS-CoV-1
    • Protein or peptide: ORF3a protein
    • Protein or peptide: Apolipoprotein A-IApolipoprotein AI
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Ligand: water
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 / phospholipid homeostasis / high-density lipoprotein particle / triglyceride homeostasis / regulation of Cdc42 protein signal transduction / cholesterol transport / chemorepellent activity / HDL remodeling / cholesterol efflux / 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
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
Apolipoprotein A-I / ORF3a protein
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.08 Å
AuthorsKern DM / Hoel CM / Kotecha A / Brohawn SG
Funding support United States, 2 items
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
CitationJournal: 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, 2020-
Header (metadata) releaseNov 18, 2020-
Map releaseNov 18, 2020-
UpdateAug 31, 2022-
Current statusAug 31, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7kjr
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22898.png

Downloads & links

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Map

FileDownload / File: emd_22898.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened and locally filtered map
Voxel sizeX=Y=Z: 0.727 Å
Density
Contour LevelBy AUTHOR: 0.7 / Movie #1: 0.6
Minimum - Maximum-2.3187268 - 3.9089978
Average (Standard dev.)0.0021007755 (±0.067432046)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 218.1 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.7270.7270.727
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z218.100218.100218.100
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ600600600
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-2.3193.9090.002

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Supplemental data

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Additional map: Density modified map

Fileemd_22898_additional_1.map
AnnotationDensity modified map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_22898_half_map_1.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_22898_half_map_2.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : SARS-CoV-2 protein 3A in lipid nanodiscs

EntireName: SARS-CoV-2 protein 3A in lipid nanodiscsSARS-CoV-1
Components
  • Complex: SARS-CoV-2 protein 3A in lipid nanodiscsSARS-CoV-1
    • Protein or peptide: ORF3a protein
    • Protein or peptide: Apolipoprotein A-IApolipoprotein AI
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Ligand: water

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Supramolecule #1: SARS-CoV-2 protein 3A in lipid nanodiscs

SupramoleculeName: SARS-CoV-2 protein 3A in lipid nanodiscs / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
Molecular weightTheoretical: 62 KDa

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Macromolecule #1: ORF3a protein

MacromoleculeName: ORF3a protein / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 32.165902 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDLFMRIFTI GTVTLKQGEI KDATPSDFVR ATATIPIQAS LPFGWLIVGV ALLAVFQSAS KIITLKKRWQ LALSKGVHFV CNLLLLFVT VYSHLLLVAA GLEAPFLYLY ALVYFLQSIN FVRIIMRLWL CWKCRSKNPL LYDANYFLCW HTNCYDYCIP Y NSVTSSIV ...String:
MDLFMRIFTI GTVTLKQGEI KDATPSDFVR ATATIPIQAS LPFGWLIVGV ALLAVFQSAS KIITLKKRWQ LALSKGVHFV CNLLLLFVT VYSHLLLVAA GLEAPFLYLY ALVYFLQSIN FVRIIMRLWL CWKCRSKNPL LYDANYFLCW HTNCYDYCIP Y NSVTSSIV ITSGDGTTSP ISEHDYQIGG YTEKWESGVK DCVVLHSYFT SDYYQLYSTQ LSTDTGVEHV TFFIYNKIVD EP EEHVQIH TIDGSSGVVN PVMEPIYDEP TTTTSVPLSN SLEVLFQ

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Macromolecule #2: Apolipoprotein A-I

MacromoleculeName: Apolipoprotein A-I / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.647678 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: HHHHHHHDYD IPTTENLYFQ GSTFSKLREQ LGPVTQEFWD NLEKETEGLR QEMSKDLEEV KAKVQPYLDD FQKKWQEEME LYRQKVEPL RAELQEGARQ KLHELQEKLS PLGEEMRDRA RAHVDALRTH LAPYSDELRQ RLAARLEALK ENGGARLAEY H AKATEHLS ...String:
HHHHHHHDYD IPTTENLYFQ GSTFSKLREQ LGPVTQEFWD NLEKETEGLR QEMSKDLEEV KAKVQPYLDD FQKKWQEEME LYRQKVEPL RAELQEGARQ KLHELQEKLS PLGEEMRDRA RAHVDALRTH LAPYSDELRQ RLAARLEALK ENGGARLAEY H AKATEHLS TLSEKAKPAL EDLRQGLLPV LESFKVSFLS ALEEYTKKLN TQ

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Macromolecule #3: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine

MacromoleculeName: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / type: ligand / ID: 3 / Number of copies: 2 / Formula: PEE
Molecular weightTheoretical: 744.034 Da
Chemical component information

ChemComp-PEE:
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE, phospholipid*YM / Discrete optimized protein energy

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 122 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.1 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMKClPotassium Chloride
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: 1 blot force 5 second wait time 3 second blot time.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Ab initio model
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 2.08 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 91218

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Atomic model buiding 1

RefinementSpace: REAL
Output model

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

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