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- EMDB-28545: Structure of SARS-CoV-2 Orf3a in plasma membrane-like environment... -

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Basic information

Entry
Database: EMDB / ID: EMD-28545
TitleStructure of SARS-CoV-2 Orf3a in plasma membrane-like environment, MSP1D1 nanodisc
Map data
Sample
  • Complex: Structure of SARS-CoV2 Orf3a in plasma membrane-like environment, MSP1D1 nanodisc
    • Protein or peptide: ORF3a protein
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
Function / homology
Function and homology information


host cell lysosome / induction by virus of host reticulophagy / Maturation of protein 3a / SARS-CoV-2 modulates autophagy / inorganic cation transmembrane transport / host cell endoplasmic reticulum / voltage-gated calcium channel complex / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / voltage-gated potassium channel complex / molecular function activator activity ...host cell lysosome / induction by virus of host reticulophagy / Maturation of protein 3a / SARS-CoV-2 modulates autophagy / inorganic cation transmembrane transport / host cell endoplasmic reticulum / voltage-gated calcium channel complex / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / voltage-gated potassium channel complex / molecular function activator activity / : / protein complex oligomerization / monoatomic ion channel activity / host cell endosome / Translation of Structural Proteins / Virion Assembly and Release / Induction of Cell-Cell Fusion / Attachment and Entry / host cell endoplasmic reticulum membrane / SARS-CoV-2 activates/modulates innate and adaptive immune responses / host cell plasma membrane / virion membrane / extracellular region / identical protein binding / plasma membrane
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.
Similarity search - Domain/homology
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsMiller AN / Houlihan PR / Matamala E / Cabezas-Bratesco D / Lee GY / Cristofori-Armstrong B / Dilan TL / Sanchez-Martinez S / Matthies D / Yan R ...Miller AN / Houlihan PR / Matamala E / Cabezas-Bratesco D / Lee GY / Cristofori-Armstrong B / Dilan TL / Sanchez-Martinez S / Matthies D / Yan R / Yu Z / Ren D / Brauchi SE / Clapham DE
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: bioRxiv / Year: 2022
Title: The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins.
Authors: Alexandria N Miller / Patrick R Houlihan / Ella Matamala / Deny Cabezas-Bratesco / Gi Young Lee / Ben Cristofori-Armstrong / Tanya L Dilan / Silvia Sanchez-Martinez / Doreen Matthies / Rui ...Authors: Alexandria N Miller / Patrick R Houlihan / Ella Matamala / Deny Cabezas-Bratesco / Gi Young Lee / Ben Cristofori-Armstrong / Tanya L Dilan / Silvia Sanchez-Martinez / Doreen Matthies / Rui Yan / Zhiheng Yu / Dejian Ren / Sebastian E Brauchi / David E Clapham
Abstract: The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi ...The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as a viroporin. Here we show that neither SARS-CoV-2 nor SARS-CoV-1 form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a basic aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.
History
DepositionOct 9, 2022-
Header (metadata) releaseFeb 8, 2023-
Map releaseFeb 8, 2023-
UpdateFeb 8, 2023-
Current statusFeb 8, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28545.png

Downloads & links

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Map

FileDownload / File: emd_28545.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.844 Å
Density
Contour LevelBy AUTHOR: 0.015
Minimum - Maximum-0.042586695 - 0.09119789
Average (Standard dev.)0.00012984389 (±0.0016345188)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 216.064 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_28545_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_28545_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : Structure of SARS-CoV2 Orf3a in plasma membrane-like environment,...

EntireName: Structure of SARS-CoV2 Orf3a in plasma membrane-like environment, MSP1D1 nanodisc
Components
  • Complex: Structure of SARS-CoV2 Orf3a in plasma membrane-like environment, MSP1D1 nanodisc
    • Protein or peptide: ORF3a protein
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine

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Supramolecule #1: Structure of SARS-CoV2 Orf3a in plasma membrane-like environment,...

SupramoleculeName: Structure of SARS-CoV2 Orf3a in plasma membrane-like environment, MSP1D1 nanodisc
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2

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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: 36.489445 KDa
Recombinant expressionOrganism: Homo sapiens (human)
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 TTTTSVPLGG RGLEVLFQGP GSGQLVGSGG LEGGGGWSHP QFEKGGGSGG GSG GGSWSH PQFEK

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

MacromoleculeName: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / type: ligand / ID: 2 / Number of copies: 4 / 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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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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 / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 15637 / 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: PDB ENTRY
PDB model - PDB ID:

7kjr

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 125625

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-8eqt:
Structure of SARS-CoV-2 Orf3a in plasma membrane-like environment, MSP1D1 nanodisc

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