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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 nanodiscs
    • Protein or peptide: ORF3a protein
    • Protein or peptide: Apolipoprotein A-I
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Ligand: water
KeywordsSARS-CoV-2 / coronavirus / transport protein / viral protein / viroporin / membrane protein
Function / homology
Function and homology information


host cell lysosome / induction by virus of host reticulophagy / Maturation of protein 3a / Defective ABCA1 causes TGD / high-density lipoprotein particle receptor binding / Scavenging by Class B Receptors / HDL clearance / spherical high-density lipoprotein particle / positive regulation of hydrolase activity / negative regulation of response to cytokine stimulus ...host cell lysosome / induction by virus of host reticulophagy / Maturation of protein 3a / Defective ABCA1 causes TGD / high-density lipoprotein particle receptor binding / Scavenging by Class B Receptors / HDL clearance / spherical high-density lipoprotein particle / positive regulation of hydrolase activity / negative regulation of response to cytokine stimulus / protein oxidation / regulation of intestinal cholesterol absorption / vitamin transport / cholesterol import / high-density lipoprotein particle binding / SARS-CoV-2 modulates autophagy / blood vessel endothelial cell migration / ABC transporters in lipid homeostasis / negative regulation of heterotypic cell-cell adhesion / apolipoprotein receptor binding / apolipoprotein A-I receptor binding / negative regulation of cytokine production involved in immune response / negative regulation of cell adhesion molecule production / HDL assembly / negative regulation of very-low-density lipoprotein particle remodeling / peptidyl-methionine modification / phosphatidylcholine biosynthetic process / glucocorticoid metabolic process / acylglycerol homeostasis / Chylomicron remodeling / cellular response to lipoprotein particle stimulus / phosphatidylcholine-sterol O-acyltransferase activator activity / positive regulation of phospholipid efflux / Chylomicron assembly / positive regulation of cholesterol metabolic process / lipid storage / high-density lipoprotein particle clearance / chylomicron / high-density lipoprotein particle remodeling / phospholipid efflux / phospholipid homeostasis / cholesterol transfer activity / chemorepellent activity / reverse cholesterol transport / high-density lipoprotein particle assembly / very-low-density lipoprotein particle / low-density lipoprotein particle / lipoprotein biosynthetic process / positive regulation of CoA-transferase activity / cholesterol transport / high-density lipoprotein particle / regulation of Cdc42 protein signal transduction / triglyceride homeostasis / inorganic cation transmembrane transport / HDL remodeling / endothelial cell proliferation / cholesterol efflux / Scavenging by Class A Receptors / voltage-gated calcium channel complex / negative regulation of interleukin-1 beta production / host cell endoplasmic reticulum / cholesterol binding / adrenal gland development / negative chemotaxis / positive regulation of Rho protein signal transduction / cholesterol biosynthetic process / monoatomic ion channel activity / endocytic vesicle / negative regulation of tumor necrosis factor-mediated signaling pathway / positive regulation of cholesterol efflux / Scavenging of heme from plasma / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / voltage-gated potassium channel complex / Retinoid metabolism and transport / positive regulation of substrate adhesion-dependent cell spreading / positive regulation of phagocytosis / positive regulation of stress fiber assembly / endocytic vesicle lumen / heat shock protein binding / cholesterol metabolic process / cholesterol homeostasis / molecular function activator activity / integrin-mediated signaling pathway / Post-translational protein phosphorylation / Heme signaling / regulation of protein phosphorylation / 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) / extracellular vesicle / Platelet degranulation / amyloid-beta binding / host cell endosome / cytoplasmic vesicle / collagen-containing extracellular matrix / secretory granule lumen / blood microparticle / Translation of Structural Proteins
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
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-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing 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
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 300 pix.
= 218.1 Å		0.73 Å/pix.
x 300 pix.
= 218.1 Å		0.73 Å/pix.
x 300 pix.
= 218.1 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.727 Å
Density

Histogram

Histogram (log scale)
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

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Slices (1/2)
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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 nanodiscs
Components
  • Complex: SARS-CoV-2 protein 3A in lipid nanodiscs
    • Protein or peptide: ORF3a protein
    • Protein or peptide: Apolipoprotein A-I
  • 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
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

UniProtKB: ORF3a protein

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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

UniProtKB: Apolipoprotein A-I

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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

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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

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

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Image processing

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

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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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