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- EMDB-28544: Structure of SARS-CoV-1 Orf3a in late endosome/lysosome-like envi... -

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Basic information

Entry
Database: EMDB / ID: EMD-28544
TitleStructure of SARS-CoV-1 Orf3a in late endosome/lysosome-like environment, MSP1D1 nanodisc
Map data
Sample
  • Complex: Structure of SARS-CoV-1 Orf3a in late endosome/lysosome-like environment, MSP1D1 nanodisc
    • Protein or peptide: ORF3a protein
    • Protein or peptide: Apolipoprotein A-I
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
KeywordsMembrane protein / SARS-CoV / SARS-CoV-2 / VIRAL PROTEIN
Function / homology
Function and homology information


Maturation of protein 3a / Defective ABCA1 causes TGD / high-density lipoprotein particle receptor binding / Scavenging by Class B Receptors / HDL clearance / apolipoprotein receptor binding / spherical high-density lipoprotein particle / positive regulation of hydrolase activity / negative regulation of response to cytokine stimulus / protein oxidation ...Maturation of protein 3a / Defective ABCA1 causes TGD / high-density lipoprotein particle receptor binding / Scavenging by Class B Receptors / HDL clearance / apolipoprotein receptor binding / 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 / blood vessel endothelial cell migration / ABC transporters in lipid homeostasis / negative regulation of heterotypic cell-cell adhesion / Microbial modulation of RIPK1-mediated regulated necrosis / 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 / Translation of Structural Proteins / SARS-CoV-1-mediated effects on programmed cell death / Virion Assembly and Release / 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 / phospholipid homeostasis / high-density lipoprotein particle remodeling / phospholipid efflux / cholesterol transfer activity / chemorepellent activity / reverse cholesterol transport / high-density lipoprotein particle assembly / cholesterol transport / very-low-density lipoprotein particle / low-density lipoprotein particle / lipoprotein biosynthetic process / positive regulation of CoA-transferase activity / high-density lipoprotein particle / regulation of Cdc42 protein signal transduction / triglyceride homeostasis / inorganic cation transmembrane transport / HDL remodeling / endothelial cell proliferation / Scavenging by Class A Receptors / cholesterol efflux / voltage-gated calcium channel complex / negative regulation of interleukin-1 beta production / cholesterol binding / negative chemotaxis / adrenal gland development / positive regulation of Rho protein signal transduction / cholesterol biosynthetic process / host cell Golgi membrane / endocytic vesicle / monoatomic ion channel activity / negative regulation of tumor necrosis factor-mediated signaling pathway / positive regulation of cholesterol efflux / Scavenging of heme from plasma / voltage-gated potassium channel complex / Retinoid metabolism and transport / positive regulation of substrate adhesion-dependent cell spreading / SARS-CoV-1 targets host intracellular signalling and regulatory pathways / positive regulation of phagocytosis / heat shock protein binding / positive regulation of stress fiber assembly / endocytic vesicle lumen / Attachment and Entry / cholesterol metabolic process / cholesterol homeostasis / 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 / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / SARS-CoV-1 activates/modulates innate immune responses / extracellular vesicle / Platelet degranulation / amyloid-beta binding / cytoplasmic vesicle / collagen-containing extracellular matrix / secretory granule lumen / blood microparticle / early endosome
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 speciesHomo sapiens (human) / Severe acute respiratory syndrome coronavirus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 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-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28544.png

Downloads & links

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Map

FileDownload / File: emd_28544.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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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

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.844 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.018
Minimum - Maximum-0.045289513 - 0.096237026
Average (Standard dev.)0.000116909934 (±0.0018406784)
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_28544_half_map_1.map
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Half map: #1

Fileemd_28544_half_map_2.map
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Sample components

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

EntireName: Structure of SARS-CoV-1 Orf3a in late endosome/lysosome-like environment, MSP1D1 nanodisc
Components
  • Complex: Structure of SARS-CoV-1 Orf3a in late endosome/lysosome-like environment, MSP1D1 nanodisc
    • Protein or peptide: ORF3a protein
    • Protein or peptide: Apolipoprotein A-I
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine

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

SupramoleculeName: Structure of SARS-CoV-1 Orf3a in late endosome/lysosome-like environment, MSP1D1 nanodisc
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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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
Molecular weightTheoretical: 36.268219 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDLFMRFFTL GSITAQPVKI DNASPASTVH ATATIPLQAS LPFGWLVIGV AFLAVFQSAT KIIALNKRWQ LALYKGFQFI CNLLLLFVT IYSHLLLVAA GMEAQFLYLY ALIYFLQCIN ACRIIMRCWL CWKCKSKNPL LYDANYFVCW HTHNYDYCIP Y NSVTDTIV ...String:
MDLFMRFFTL GSITAQPVKI DNASPASTVH ATATIPLQAS LPFGWLVIGV AFLAVFQSAT KIIALNKRWQ LALYKGFQFI CNLLLLFVT IYSHLLLVAA GMEAQFLYLY ALIYFLQCIN ACRIIMRCWL CWKCKSKNPL LYDANYFVCW HTHNYDYCIP Y NSVTDTIV VTEGDGISTP KLKEDYQIGG YSEDRHSGVK DYVVVHGYFT EVYYQLESTQ ITTDTGIENA TFFIFNKLVK DP PNVQIHT IDGSSGVANP AMDPIYDEPT TTTSVPLGGR GLEVLFQGPG SGQLVGSGGL EGGGGWSHPQ FEKGGGSGGG SGG GSWSHP QFEK

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: 24.704729 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GHHHHHHHDY DIPTTENLYF QGSTFSKLRE QLGPVTQEFW DNLEKETEGL RQEMSKDLEE VKAKVQPYLD DFQKKWQEEM ELYRQKVEP LRAELQEGAR QKLHELQEKL SPLGEEMRDR ARAHVDALRT HLAPYSDELR QRLAARLEAL KENGGARLAE Y HAKATEHL ...String:
GHHHHHHHDY DIPTTENLYF QGSTFSKLRE QLGPVTQEFW DNLEKETEGL RQEMSKDLEE VKAKVQPYLD DFQKKWQEEM ELYRQKVEP LRAELQEGAR QKLHELQEKL SPLGEEMRDR ARAHVDALRT HLAPYSDELR QRLAARLEAL KENGGARLAE Y HAKATEHL STLSEKAKPA LEDLRQGLLP VLESFKVSFL SALEEYTKKL NTQ

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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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

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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: 1 / Number real images: 13970 / 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 reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 162607
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-8eqs:
Structure of SARS-CoV-1 Orf3a in late endosome/lysosome-like environment, MSP1D1 nanodisc

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