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- PDB-9oal: Cryo-EM structure of EBV gB prefusion construct C3-GT -

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Basic information

Entry
Database: PDB / ID: 9oal
TitleCryo-EM structure of EBV gB prefusion construct C3-GT
ComponentsEnvelope glycoprotein B
KeywordsVIRAL PROTEIN / fusion protein / engineered protein / stabilized protein / prefusion protein / gB / glycoprotein B
Function / homology
Function and homology information


host cell Golgi membrane / host cell endosome membrane / viral envelope / symbiont entry into host cell / virion attachment to host cell / host cell plasma membrane / virion membrane / membrane
Similarity search - Function
: / Herpesvirus Glycoprotein B / Herpesvirus Glycoprotein B, PH-like domain 1 / Herpesvirus Glycoprotein B, PH-like domain 2 / Herpesvirus Glycoprotein B, PH-like domain 2 superfamily / Herpesvirus Glycoprotein B ectodomain / Herpesvirus Glycoprotein B / Herpesvirus Glycoprotein B PH-like domain
Similarity search - Domain/homology
Envelope glycoprotein B
Similarity search - Component
Biological specieshuman gammaherpesvirus 4 (Epstein-Barr virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsMcCool, R.S. / McLellan, J.S.
Funding support United States, 1items
OrganizationGrant numberCountry
Other private United States
CitationJournal: Nat Commun / Year: 2026
Title: Structure and immunogenicity of an engineered soluble prefusion-stabilized EBV gB antigen.
Authors: Ryan S McCool / Cory M Acreman / Abigail E Powell / Sofia I Picucci / Daniel J Stieh / Chia-Wei Chou / Jeremy Huynh / Hannah Caruso / Soyoon Park / Jessica O'Rear / Jui-Lin Chen / Brad A ...Authors: Ryan S McCool / Cory M Acreman / Abigail E Powell / Sofia I Picucci / Daniel J Stieh / Chia-Wei Chou / Jeremy Huynh / Hannah Caruso / Soyoon Park / Jessica O'Rear / Jui-Lin Chen / Brad A Palanski / Patrick O Byrne / Madeline R Sponholtz / Jeongryeol Kim / Julie E Ledgerwood / Payton A-B Weidenbacher / Jason S McLellan /
Abstract: Epstein-Barr virus (EBV), the causative agent of mononucleosis, is linked to over 140,000 annual cancer-related deaths globally and increases the risk of multiple sclerosis by up to 32-fold. As a ...Epstein-Barr virus (EBV), the causative agent of mononucleosis, is linked to over 140,000 annual cancer-related deaths globally and increases the risk of multiple sclerosis by up to 32-fold. As a herpesvirus, EBV establishes lifelong infection, and over 90% of U.S. adults are EBV-seropositive. Despite its significant disease burden, no approved EBV vaccines or therapeutics exist. Among EBV envelope glycoproteins, the fusion protein (gB) is strictly required for epithelial and B cell infection. Here, using a combination of AlphaFold-guided modeling, rational design, and ThermoMPNN-informed optimization, we engineer a stabilized prefusion gB variant, C3-GT. This construct incorporates two inter-protomeric disulfide bonds and three cavity-filling substitutions, resulting in a melting temperature of 54 °C. Cryo-EM analysis of this construct reveals the prefusion structure of EBV gB, providing insights into the structural transitions required to adopt the postfusion conformation. Murine immunizations and depletion studies with human sera suggest a trend toward improved functional immunogenicity of C3-GT compared to postfusion gB. Collectively, these studies define engineering principles to stabilize class III fusion proteins, provide reagents to interrogate the human antibody response to EBV gB, and lay a foundation for further studies to develop EBV gB-based vaccine candidates.
History
DepositionApr 21, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 31, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Envelope glycoprotein B
B: Envelope glycoprotein B
C: Envelope glycoprotein B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)259,6796
Polymers257,9203
Non-polymers1,7603
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Envelope glycoprotein B / gB


Mass: 85973.250 Da / Num. of mol.: 3
Mutation: I89C, W112H, Y113R, W193R, L194V, I195E, W196A, A175C, D220E, H316I, D320Q, S325L, R428G, R429G, R439S, R431G, R432G, L628GCG, E634C
Source method: isolated from a genetically manipulated source
Details: C-terminally truncated recombinant ectodomain construct
Source: (gene. exp.) human gammaherpesvirus 4 (Epstein-Barr virus)
Strain: M81 / Gene: gB, BALF4 / Cell line (production host): Hek293F / Production host: Homo sapiens (human) / References: UniProt: P0C762
#2: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
Has ligand of interestN
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: Prefusion-stabilized Epstein-Barr virus glycoprotein B
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.251 MDa / Experimental value: NO
Source (natural)Organism: human gammaherpesvirus 4 (Epstein-Barr virus) / Strain: M81
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
Details: 2 mM Tris (pH 8.0), 200 mM NaCl, 2 mM CaCl2, and 0.02% (w/v) sodium azide (SEC buffer). CHAPS was added immediately before freezing to a final concentration of 2 mM (0.25x CMC or 0.123% w/v).
Buffer component
IDConc.NameFormulaBuffer-ID
12 mMTris base1
2200 mMsodium chlorideNaCl1
32 mMcalcium chlorideCaCl21
40.02 % (w/v)sodium azideNaN31
51
SpecimenConc.: 6.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 150000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 49 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4541

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.5.3particle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
7PHENIXmodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13Cootmodel refinement
14ISOLDEmodel refinement
Image processingDetails: Prefusion stabilized Epstein-Barr virus envelope glycoprotein B
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2178814
Details: Particle picking was performed initially using blob picker. After initial, on-the-fly processing in CryoSparc Live, templates were generated for subsequent template-based picking.
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 162928 / Symmetry type: POINT
Atomic model buildingDetails: Template-based, PDB: 7KDP / Source name: AlphaFold / Type: in silico model

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