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- EMDB-70288: Cryo-EM structure of EBV gB prefusion construct C3-GT -

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Basic information

Entry
Database: EMDB / ID: EMD-70288
TitleCryo-EM structure of EBV gB prefusion construct C3-GT
Map dataHalf map B. Vop flipped and resampled in ChimeraX to improve view upon open.
Sample
  • Complex: Prefusion-stabilized Epstein-Barr virus glycoprotein B
    • Protein or peptide: Envelope glycoprotein B
Keywordsfusion protein / VIRAL 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsMcCool RS / McLellan JS
Funding support United States, 1 items
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, 2025-
Header (metadata) releaseDec 31, 2025-
Map releaseDec 31, 2025-
UpdateFeb 11, 2026-
Current statusFeb 11, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_70288.png

Downloads & links

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Map

FileDownload / File: emd_70288.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHalf map B. Vop flipped and resampled in ChimeraX to improve view upon open.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.93 Å/pix.
x 384 pix.
= 358.272 Å		0.93 Å/pix.
x 384 pix.
= 358.272 Å		0.93 Å/pix.
x 384 pix.
= 358.272 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.933 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.013580838 - 1.3917648
Average (Standard dev.)0.00057233695 (±0.0123039195)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 358.272 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_70288_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: #1

Fileemd_70288_additional_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B. Vop flipped and resampled in...

Fileemd_70288_half_map_1.map
AnnotationHalf map B. Vop flipped and resampled in ChimeraX to improve view upon open.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B. Vop flipped and resampled in...

Fileemd_70288_half_map_2.map
AnnotationHalf map B. Vop flipped and resampled in ChimeraX to improve view upon open.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Prefusion-stabilized Epstein-Barr virus glycoprotein B

EntireName: Prefusion-stabilized Epstein-Barr virus glycoprotein B
Components
  • Complex: Prefusion-stabilized Epstein-Barr virus glycoprotein B
    • Protein or peptide: Envelope glycoprotein B

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Supramolecule #1: Prefusion-stabilized Epstein-Barr virus glycoprotein B

SupramoleculeName: Prefusion-stabilized Epstein-Barr virus glycoprotein B
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: human gammaherpesvirus 4 (Epstein-Barr virus) / Strain: M81
Molecular weightTheoretical: 251 KDa

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Macromolecule #1: Envelope glycoprotein B

MacromoleculeName: Envelope glycoprotein B / type: protein_or_peptide / ID: 1
Details: C-terminally truncated recombinant ectodomain construct
Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: human gammaherpesvirus 4 (Epstein-Barr virus) / Strain: M81
Molecular weightTheoretical: 85.97325 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MTRRRVLSVV VLLAALACRL GAQTPEQPAP PATTVQPTAT RQQTSFPFRV CELSSHGDLF RFSSDIQCPS FGTRENHTEG LLMVFKDNC IPYSFKVRSY TKIVTNILIY NGHRADSVTN RHEEKFSVES YETDQMDTIY QCYNAVKMTK DGLTRVYVDR D GVNITVNL ...String:
MTRRRVLSVV VLLAALACRL GAQTPEQPAP PATTVQPTAT RQQTSFPFRV CELSSHGDLF RFSSDIQCPS FGTRENHTEG LLMVFKDNC IPYSFKVRSY TKIVTNILIY NGHRADSVTN RHEEKFSVES YETDQMDTIY QCYNAVKMTK DGLTRVYVDR D GVNITVNL KPTGGLCNGV RRYASQTELY DAPGRVEATY RTRTTVNCLI TDMMAKSNSP FEFFVTTTGQ TVEMSPFYDG KN TETFHER ADSFHVRTNY KIVDYDNRGT NPQGERRAFL DKGTYTLSWK LENRTAYCPL QHWQTFDSTI ATETGKSIIF VTQ EGTSLF VTNTTVGIEL PDAFKCIEEQ VNKTMHEKYE AVQDRYTKGQ EAITYFITSG GLLLAWLPLT PRSLATVKNL TELT TPTSS PPSSPSPPAP PAARGSTSAA VLGGSGGNAG NATTPVPPAA PGKSLGTLNN PATVQIQFAY DSLRRQINRM LGDLA RAWC LEQKRQNMVL RELTKINPTT VMSSIYGKAV AAKRLGDVIS VSQCVPVNQA TVTLRKSMRV PGSETMCYSR PLVSFS FIN DTKTYEGQLG TDNEIFLTKK MTEVCQATSQ YYFQSGNEIH VYNDYHHFKT IELDGIATLQ TFISGCGNTS LICNIDF AS LELYSRDEQR ASNVFDLEGI FREYNFQAQN IAGLRKDLDN AVSNGRNQGG SGYIPEAPRD GQAYVRKDGE WVLLSTFL G RAAASSLEVL FQGPGHHHHH HHHSAWSHPQ FEKGGGSGGG GSGGSAWSHP QFEK

UniProtKB: Envelope glycoprotein B

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration6.2 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
2.0 mMTris base
200.0 mMsodium chlorideNaCl
2.0 mMcalcium chlorideCaCl2
0.02 % (w/v)sodium azideNaN3

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).
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 4541 / Average electron dose: 49.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 150000

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Image processing

DetailsPrefusion stabilized Epstein-Barr virus envelope glycoprotein B
Particle selectionNumber 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.
CTF correctionSoftware - Name: cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
In silico model: Template-based AlphaFold2 model. PDB 7KDP was used as the template.
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 162928
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 5 / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model / Details: Template-based, PDB: 7KDP
Output model

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

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