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Yorodumi- PDB-8equ: Structure of SARS-CoV-2 Orf3a in late endosome/lysosome-like envi... -
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-Basic information
Entry | Database: PDB / ID: 8equ | ||||||
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Title | Structure of SARS-CoV-2 Orf3a in late endosome/lysosome-like environment, Saposin A nanodisc | ||||||
Components |
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Keywords | VIRAL 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 / 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 / regulation of lipid metabolic process / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / voltage-gated potassium channel complex / enzyme activator activity / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / lysosomal lumen / Peptide ligand-binding receptors / regulation of autophagy / molecular function activator activity / cytoplasmic side of plasma membrane / 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 / protease binding / 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 | ||||||
Biological species | Severe acute respiratory syndrome coronavirus 2 Homo sapiens (human) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å | ||||||
Authors | 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. ...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
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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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
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PDBx/mmCIF format | 8equ.cif.gz | 140.3 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8equ.ent.gz | 109.3 KB | Display | PDB format |
PDBx/mmJSON format | 8equ.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8equ_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 8equ_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 8equ_validation.xml.gz | 32.2 KB | Display | |
Data in CIF | 8equ_validation.cif.gz | 45.8 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/eq/8equ ftp://data.pdbj.org/pub/pdb/validation_reports/eq/8equ | HTTPS FTP |
-Related structure data
Related structure data | 28546MC 8eqjC 8eqsC 8eqtC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | 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 | 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 | 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 | Has ligand of interest | N | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Structure of SARS-CoV-2 Orf3a in late endosome/lysosome-like environment, Saposin A nanodisc Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT |
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Source (natural) | Organism: Severe acute respiratory syndrome coronavirus 2 |
Source (recombinant) | Organism: Homo sapiens (human) |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm |
Image recording | Electron 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 optics | Energyfilter name: GIF Bioquantum |
-Processing
Software | Name: PHENIX / Version: 1.19.2_4158: / Classification: refinement | ||||||||||||||||||||||||
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EM software |
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CTF correction | Type: NONE | ||||||||||||||||||||||||
Symmetry | Point symmetry: C2 (2 fold cyclic) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 135280 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Protocol: AB INITIO MODEL | ||||||||||||||||||||||||
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