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- EMDB-28546: Structure of SARS-CoV-2 Orf3a in late endosome/lysosome-like envi... -

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Basic information

Entry
Database: EMDB / ID: EMD-28546
TitleStructure of SARS-CoV-2 Orf3a in late endosome/lysosome-like environment, Saposin A nanodisc
Map data
Sample
  • Complex: Structure of SARS-CoV-2 Orf3a in late endosome/lysosome-like environment, Saposin A nanodisc
    • Protein or peptide: ORF3a protein
    • Protein or peptide: Saposin-A
    • Protein or peptide: Saposin A, polyalanine model
  • 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 / positive regulation of beta-galactosidase activity / ganglioside GM1 transport to membrane / ganglioside GM2 binding / ganglioside GM3 binding / ganglioside GP1c binding / ganglioside GM1 binding / ganglioside GT1b binding ...host cell lysosome / induction by virus of host reticulophagy / Maturation of protein 3a / positive regulation of beta-galactosidase activity / ganglioside GM1 transport to membrane / ganglioside GM2 binding / ganglioside GM3 binding / ganglioside GP1c binding / ganglioside GM1 binding / ganglioside GT1b binding / SARS-CoV-2 modulates autophagy / sphingolipid metabolic process / prostate gland growth / epithelial cell differentiation involved in prostate gland development / Glycosphingolipid catabolism / inorganic cation transmembrane transport / voltage-gated calcium channel complex / lysosomal transport / azurophil granule membrane / host cell endoplasmic reticulum / monoatomic ion channel activity / regulation of lipid metabolic process / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / voltage-gated potassium channel complex / enzyme activator activity / lysosomal lumen / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / Peptide ligand-binding receptors / molecular function activator activity / regulation of autophagy / phospholipid binding / cytoplasmic side of plasma membrane / late endosome / Platelet degranulation / host cell endosome / G alpha (i) signalling events / scaffold protein binding / protease binding / collagen-containing extracellular matrix / Translation of Structural Proteins / Virion Assembly and Release / Induction of Cell-Cell Fusion / Attachment and Entry / lysosome / host cell endoplasmic reticulum membrane / lysosomal membrane / intracellular membrane-bounded organelle / Neutrophil degranulation / SARS-CoV-2 activates/modulates innate and adaptive immune responses / host cell plasma membrane / virion membrane / protein homodimerization activity / extracellular space / extracellular exosome / 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. / Saposin, chordata / Saposin A-type domain / Saposin / : ...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. / Saposin, chordata / Saposin A-type domain / Saposin / : / Saposin A-type domain / Saposin A-type domain profile. / Saposin/surfactant protein-B A-type DOMAIN / Saposin-like type B, region 1 / Saposin-like type B, region 1 / Saposin B type, region 2 / Saposin-like type B, region 2 / Saposin (B) Domains / Saposin B type domain / Saposin-like / Saposin B type domain profile.
Similarity search - Domain/homology
Prosaposin / ORF3a protein
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 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_28546.png

Downloads & links

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Map

FileDownload / File: emd_28546.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 256 pix.
= 216.064 Å		0.84 Å/pix.
x 256 pix.
= 216.064 Å		0.84 Å/pix.
x 256 pix.
= 216.064 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.844 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.012
Minimum - Maximum-0.074140586 - 0.15199354
Average (Standard dev.)0.00014781288 (±0.0025827773)
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: #1

Fileemd_28546_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

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Histogram (log scale)

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Half map: #2

Fileemd_28546_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Structure of SARS-CoV-2 Orf3a in late endosome/lysosome-like envi...

EntireName: Structure of SARS-CoV-2 Orf3a in late endosome/lysosome-like environment, Saposin A nanodisc
Components
  • Complex: Structure of SARS-CoV-2 Orf3a in late endosome/lysosome-like environment, Saposin A nanodisc
    • Protein or peptide: ORF3a protein
    • Protein or peptide: Saposin-A
    • Protein or peptide: Saposin A, polyalanine model
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine

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Supramolecule #1: Structure of SARS-CoV-2 Orf3a in late endosome/lysosome-like envi...

SupramoleculeName: Structure of SARS-CoV-2 Orf3a in late endosome/lysosome-like environment, Saposin A nanodisc
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#3
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: Saposin-A

MacromoleculeName: Saposin-A / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.677292 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MGGHHHHHHS SGVDLGTENL YFQSMSLPCD ICKDVVTAAG DMLKDNATEE EILVYLEKTC DWLPKPNMSA SCKEIVDSYL PVILDIIKG EMSRPGEVCS ALNLCES

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Macromolecule #3: Saposin A, polyalanine model

MacromoleculeName: Saposin A, polyalanine model / type: protein_or_peptide / ID: 3 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 6.741301 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) ...String:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)

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Macromolecule #4: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine

MacromoleculeName: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / type: ligand / ID: 4 / 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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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
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Number real images: 15946 / 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 / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
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

Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 135280
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-8equ:
Structure of SARS-CoV-2 Orf3a in late endosome/lysosome-like environment, Saposin A nanodisc

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