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- PDB-8equ: Structure of SARS-CoV-2 Orf3a in late endosome/lysosome-like envi... -

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Entry
Database: PDB / ID: 8equ
TitleStructure of SARS-CoV-2 Orf3a in late endosome/lysosome-like environment, Saposin A nanodisc
Components
  • ORF3a protein
  • Saposin A, polyalanine model
  • Saposin-A
KeywordsVIRAL PROTEIN / Membrane protein / SARS-CoV-2
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 / SARS-CoV-2 modulates autophagy ...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 / SARS-CoV-2 modulates autophagy / ganglioside GT1b binding / prostate gland growth / sphingolipid metabolic process / Glycosphingolipid catabolism / epithelial cell differentiation involved in prostate gland development / inorganic cation transmembrane transport / lysosomal transport / host cell endoplasmic reticulum / azurophil granule membrane / voltage-gated calcium channel complex / regulation of lipid metabolic process / enzyme activator activity / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / voltage-gated potassium channel complex / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / lysosomal lumen / Peptide ligand-binding receptors / regulation of autophagy / molecular function activator activity / phospholipid binding / : / late endosome / Platelet degranulation / protein complex oligomerization / monoatomic ion channel activity / host cell endosome / G alpha (i) signalling events / scaffold protein binding / collagen-containing extracellular matrix / Translation of Structural Proteins / Virion Assembly and Release / Induction of Cell-Cell Fusion / protease binding / 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 / Saposin A-type domain ...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
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / Prosaposin / ORF3a protein
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsMiller, A.N. / Houlihan, P.R. / Matamala, E. / Cabezas-Bratesco, D. / Lee, G.Y. / Cristofori-Armstrong, B. / Dilan, T.L. / Sanchez-Martinez, S. / Matthies, D. / Yan, R. ...Miller, A.N. / Houlihan, P.R. / Matamala, E. / Cabezas-Bratesco, D. / Lee, G.Y. / Cristofori-Armstrong, B. / Dilan, T.L. / Sanchez-Martinez, S. / Matthies, D. / Yan, R. / Yu, Z. / Ren, D. / Brauchi, S.E. / Clapham, D.E.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
Citation
Journal: Elife / Year: 2023
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 viroporins. Here, we show that neither SARS-CoV-2 nor SARS-CoV-1 Orf3a 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 positively charged 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's ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.
#1: Journal: 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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 8, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: ORF3a protein
B: ORF3a protein
C: Saposin-A
D: Saposin A, polyalanine model
E: Saposin A, polyalanine model
F: Saposin-A
G: Saposin A, polyalanine model
H: Saposin A, polyalanine model
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,78710
Polymers123,2998
Non-polymers1,4882
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: cross-linking
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ORF3a protein / ORF3a / Accessory protein 3a / Protein 3a / Protein U274 / Protein X1


Mass: 36489.445 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: Homo sapiens (human) / References: UniProt: P0DTC3
#2: Protein Saposin-A / Protein A


Mass: 11677.292 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PSAP, GLBA, SAP1 / Production host: Escherichia coli (E. coli) / References: UniProt: P07602
#3: Protein
Saposin A, polyalanine model


Mass: 6741.301 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#4: Chemical ChemComp-PEE / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE / Discrete optimized protein energy


Mass: 744.034 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C41H78NO8P / Comment: DOPE, phospholipid*YM
Has ligand of interestN

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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: Structure of SARS-CoV-2 Orf3a in late endosome/lysosome-like environment, Saposin A nanodisc
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 15946
EM imaging opticsEnergyfilter name: GIF Bioquantum

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
1Topaz0.9particle selection
4CTFFIND4CTF correction
9cryoSPARC3initial Euler assignment
10RELION3.1final Euler assignment
CTF correctionType: NONE
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 135280 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0025860
ELECTRON MICROSCOPYf_angle_d0.5028042
ELECTRON MICROSCOPYf_dihedral_angle_d7.049916
ELECTRON MICROSCOPYf_chiral_restr0.0341002
ELECTRON MICROSCOPYf_plane_restr0.0031022

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