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Yorodumi- PDB-8eqj: Structure of SARS-CoV-2 Orf3a in late endosome/lysosome-like memb... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8eqj | ||||||
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Title | Structure of SARS-CoV-2 Orf3a in late endosome/lysosome-like membrane environment, MSP1D1 nanodisc | ||||||
Components | ORF3a protein | ||||||
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 / SARS-CoV-2 modulates autophagy / inorganic cation transmembrane transport / voltage-gated calcium channel complex / host cell endoplasmic reticulum / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / voltage-gated potassium channel complex / monoatomic ion channel activity ...host cell lysosome / induction by virus of host reticulophagy / Maturation of protein 3a / SARS-CoV-2 modulates autophagy / inorganic cation transmembrane transport / voltage-gated calcium channel complex / host cell endoplasmic reticulum / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / voltage-gated potassium channel complex / monoatomic ion channel activity / molecular function activator activity / cytoplasmic side of plasma membrane / 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 | ||||||
Biological species | Severe acute respiratory syndrome coronavirus 2 | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 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
-Download
PDBx/mmCIF format | 8eqj.cif.gz | 88.3 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8eqj.ent.gz | 63 KB | Display | PDB format |
PDBx/mmJSON format | 8eqj.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8eqj_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 8eqj_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 8eqj_validation.xml.gz | 26.3 KB | Display | |
Data in CIF | 8eqj_validation.cif.gz | 32.7 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/eq/8eqj ftp://data.pdbj.org/pub/pdb/validation_reports/eq/8eqj | HTTPS FTP |
-Related structure data
Related structure data | 28538MC 8eqsC 8eqtC 8equC 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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-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: Chemical | ChemComp-PEE / 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 membrane environment, MSP1D1 nanodisc Type: COMPLEX / Entity ID: #1 / 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: 14087 |
EM imaging optics | Energyfilter name: GIF Bioquantum |
-Processing
EM software |
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CTF correction | Type: NONE | ||||||||||||||||||||||||
Symmetry | Point symmetry: C2 (2 fold cyclic) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 178997 / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Protocol: AB INITIO MODEL |