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- PDB-6xdc: Cryo-EM structure of SARS-CoV-2 ORF3a -

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Basic information

Entry
Database: PDB / ID: 6xdc
TitleCryo-EM structure of SARS-CoV-2 ORF3a
ComponentsProtein 3a
KeywordsTRANSPORT PROTEIN / SARS-CoV-2 / coronavirus / viroporin / ion channel
Function / homologyProtein 3a, betacoronavirus / host cell Golgi membrane / virion / host cell plasma membrane / integral component of membrane / extracellular region / Protein 3a
Function and homology information
Biological speciesSevere acute respiratory syndrome coronavirus 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsKern, D.M. / Hoel, C.M. / Brohawn, S.G.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM128263 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM123496 United States
CitationJournal: bioRxiv / Year: 2020
Title: Cryo-EM structure of the SARS-CoV-2 3a ion channel in lipid nanodiscs.
Authors: David M Kern / Ben Sorum / Christopher M Hoel / Savitha Sridharan / Jonathan P Remis / Daniel B Toso / Stephen G Brohawn
Abstract: SARS-CoV-2 encodes three putative ion channels: E, 8a, and 3a. In related SARS-CoV-1, 3a is implicated in viral release, inflammasome activation, and cell death and its deletion reduces viral titer ...SARS-CoV-2 encodes three putative ion channels: E, 8a, and 3a. In related SARS-CoV-1, 3a is implicated in viral release, inflammasome activation, and cell death and its deletion reduces viral titer and morbidity in animal models, suggesting 3a-targeted therapeutics could treat SARS and COVID-19. However, the structural basis for the function of 3a is unknown. Here, we show that SARS-CoV-2 forms large conductance cation channels and present cryo-EM structures of dimeric and tetrameric SARS-CoV-2 3a in lipid nanodiscs. 3a adopts a novel fold and is captured in a closed or inactivated state. A narrow bifurcated exterior pore precludes conduction and leads to a large polar cavity open to the cytosol. 3a function is conserved in a common variant among circulating SARS-CoV-2 that alters the channel pore. We identify 3a-like proteins in and that infect bats and humans, suggesting therapeutics targeting 3a could treat a range of coronaviral diseases.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJun 10, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 17, 2020Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Protein 3a
B: Protein 3a


Theoretical massNumber of molelcules
Total (without water)64,3322
Polymers64,3322
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area5110 Å2
ΔGint-45 kcal/mol
Surface area19840 Å2

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Components

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


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

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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: ORF3a in MSPE3D1 nanodisc / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT
Molecular weightValue: 0.064 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 mMHEPES1
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
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: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5 sec. / Electron dose: 50.675 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2595 / Details: 50 frames per image
Image scansWidth: 11520 / Height: 8184

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.17.1_3660refinement
PHENIX1.17.1_3660refinement
EM software
IDNameVersionCategory
1Topazparticle selection
4CTFFIND4.1CTF correction
7Coot0.9-premodel fitting
9cryoSPARC2initial Euler assignment
10cryoSPARC2final Euler assignment
11cryoSPARCclassification
12cryoSPARC23D reconstruction
13PHENIX1.17.1-3660model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4134279
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 185871 / Symmetry type: POINT
Atomic model buildingB value: 108.61 / Protocol: OTHER / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 108.61 Å2
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00593244
ELECTRON MICROSCOPYf_angle_d0.78324424
ELECTRON MICROSCOPYf_chiral_restr0.0619502
ELECTRON MICROSCOPYf_plane_restr0.0077528
ELECTRON MICROSCOPYf_dihedral_angle_d18.00911098

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