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- PDB-8w1a: Cryo-EM Structure of KSHV ORF74 Apo Dimer at 2.8A -

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Basic information

Entry
Database: PDB / ID: 8w1a
TitleCryo-EM Structure of KSHV ORF74 Apo Dimer at 2.8A
Componentsviral G-protein coupled receptor
KeywordsVIRAL PROTEIN / KSHV vGPCR / Viral GPCR / KSHV ORF74 / ORF74 Apo / ORF74 Inactive
Function / homology
Function and homology information


C-C chemokine receptor activity / C-C chemokine binding / cell chemotaxis / calcium-mediated signaling / positive regulation of cytosolic calcium ion concentration / immune response / host cell plasma membrane / membrane
Similarity search - Function
: / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family)
Similarity search - Domain/homology
viral G-protein coupled receptor
Similarity search - Component
Biological speciesHuman gammaherpesvirus 8
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.89 Å
AuthorsSahoo, B. / Seo, H.D. / Dai, X. / Jung, J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA251275 United States
CitationJournal: Nat Commun / Year: 2025
Title: Structural basis for ligand promiscuity and high signaling activity of Kaposi's Sarcoma-associated Herpesvirus-encoded GPCR.
Authors: Jun Bae Park / Bibekananda Sahoo / Amita Rani Sahoo / Dokyun Kim / Hogyu David Seo / James Bowman / Mi-Jeong Kwak / Sophia Suh / Matthias Buck / Xinghong Dai / Jae U Jung /
Abstract: Kaposi's Sarcoma-associated Herpesvirus encodes ORF74, a viral G protein-coupled receptor homologous to CXCR2, which plays a crucial role in Kaposi's Sarcoma development through its high basal ...Kaposi's Sarcoma-associated Herpesvirus encodes ORF74, a viral G protein-coupled receptor homologous to CXCR2, which plays a crucial role in Kaposi's Sarcoma development through its high basal signaling activity. Our cryoEM analysis of ORF74 in ligand-free, BRIL-fused ligand-free, and CXCL1/Gi-bound forms elucidates its ligand-independent signaling activity. A widely open, static extracellular cavity facilitates ligand promiscuity by enabling dynamic access and diverse binding modes. Structural alterations in CWxP, E/DRY, and NPxxY micro-switches stabilize the active conformation, leading to constitutive signaling. Metadynamics simulations reveal a dynamic ensemble between local switch structures corresponding to the inactive and active states, supporting spontaneous activation. CXCR2-ORF74 chimeras highlight intracellular loops 2 and 3 as key modulators of basal and agonist-induced activity. This study defines the structural basis of ORF74's ligand promiscuity, spontaneous activation, and high basal signaling, providing insights into its role in viral oncogenesis.
History
DepositionFeb 15, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 27, 2025Provider: repository / Type: Initial release
Revision 1.0Aug 27, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Aug 27, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Aug 27, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Aug 27, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Aug 27, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Aug 27, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Oct 8, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: viral G-protein coupled receptor
B: viral G-protein coupled receptor


Theoretical massNumber of molelcules
Total (without water)84,2652
Polymers84,2652
Non-polymers00
Water00
1
A: viral G-protein coupled receptor


Theoretical massNumber of molelcules
Total (without water)42,1321
Polymers42,1321
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
2
B: viral G-protein coupled receptor


Theoretical massNumber of molelcules
Total (without water)42,1321
Polymers42,1321
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein viral G-protein coupled receptor / vGPCR / Protein ORF74


Mass: 42132.266 Da / Num. of mol.: 2 / Mutation: L169W, L258V
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human gammaherpesvirus 8 / Gene: ORF74 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q98146
Has protein modificationY

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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: KSHV ORF74 (vGPCR) Apo Inverted Dimer / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.0421 MDa / Experimental value: YES
Source (natural)Organism: Human gammaherpesvirus 8
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Details: 25 mM HEPES pH 7.5, 150 mM NaCl, 0.005% (w/v) LMNG, 0.0005% (w/v) CHS, 10% Glycerol (v/v)
Buffer componentConc.: 25 mM / Name: HPEPS / Formula: C8H18N2O4S
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.89 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 697456 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0024772
ELECTRON MICROSCOPYf_angle_d0.5296502
ELECTRON MICROSCOPYf_dihedral_angle_d4.189656
ELECTRON MICROSCOPYf_chiral_restr0.035791
ELECTRON MICROSCOPYf_plane_restr0.004773

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