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- PDB-9sy5: Engineering the ADDomer Nanoparticle Vaccine Scaffold for Improve... -

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Basic information

Entry
Database: PDB / ID: 9sy5
TitleEngineering the ADDomer Nanoparticle Vaccine Scaffold for Improved Assembly and Enhanced Stability.
ComponentsPenton protein
KeywordsVIRUS LIKE PARTICLE / Adenovirus / Nanoparticle / Disulphide bond / Thermostable.
Function / homologyAdenovirus penton base protein / Adenovirus penton base protein / T=25 icosahedral viral capsid / endocytosis involved in viral entry into host cell / virion attachment to host cell / host cell nucleus / structural molecule activity / Penton protein
Function and homology information
Biological speciesChimpanzee adenovirus Y25
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.61 Å
AuthorsBalchin, G. / Berger-Schaffitzel, C. / Berger, I.
Funding support United Kingdom, European Union, 3items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC) United Kingdom
Engineering and Physical Sciences Research Council United Kingdom
European Research Council (ERC)European Union
CitationJournal: ACS Synth Biol / Year: 2026
Title: Engineering the ADDomer Nanoparticle Vaccine Scaffold for Improved Assembly and Enhanced Stability.
Authors: Georgia Balchin / Burak V Kabasakal / Alessandro Strofaldi / Sophie Hall / Charlotte Fletcher / Dora Buzas / Joshua C Bufton / Sathish K N Yadav / Dakang Shen / Frederic Garzoni / H Adrian ...Authors: Georgia Balchin / Burak V Kabasakal / Alessandro Strofaldi / Sophie Hall / Charlotte Fletcher / Dora Buzas / Joshua C Bufton / Sathish K N Yadav / Dakang Shen / Frederic Garzoni / H Adrian Bunzel / Jennifer J McManus / Christiane Schaffitzel / Imre Berger /
Abstract: Virus-like particles (VLPs) are promising platforms for next-generation vaccines due to their ability to present antigens in highly ordered, repetitive geometries emulating pathogen-associated ...Virus-like particles (VLPs) are promising platforms for next-generation vaccines due to their ability to present antigens in highly ordered, repetitive geometries emulating pathogen-associated patterns to elicit potent immune responses. The ADDomer is a synthetic dodecahedral VLP scaffold derived from the penton base protein (PBP) of human adenovirus serotype 3 (Ad3). PBP tolerates insertion of multiple antigenic epitopes in flexible surface-exposed loops, and spontaneously self-assembles into ADDomer nanoparticles, but faces limitations including incomplete assembly and susceptibility to preexisting antihuman adenovirus immunity. Here, we report two complementary engineering strategies to enhance ADDomer robustness. First, we developed a Chimpanzee adenovirus Y25-based ADDomer (CHIMPSELS) to circumvent preexisting antihuman adenovirus immunity, and introduced a point mutation to restore a motif critical for dodecahedron integrity. Second, we introduced targeted intersubunit disulfide bonds to reinforce particle assembly. High-resolution electron cryo-microscopy confirmed the formation of intact dodecahedral particles, revealing that disulfide bonds stabilize distinct conformations of the PBP N-termini. Differential scanning fluorimetry and dynamic light scattering demonstrated thermal stability and elevated aggregation onset temperatures in the disulfide-stabilized ADDomers, providing a scalable assay for screening ADDomer-based VLP constructs for vaccine development. Incorporation of validated immunogenic epitopes, including a SARS-CoV-2 receptor-binding motif segment and the Chikungunya E2EP3 peptide, demonstrated structural integrity and epitope display by the modified scaffolds. Our results establish a versatile, thermostable VLP platform with reduced susceptibility to preexisting immunity, improved particle integrity, and capacity for modular epitope presentation. This work advances the ADDomer toward practical applications in vaccine development and highlights engineering strategies that can be broadly applied to enhance the performance of protein-based VLP vaccines.
History
DepositionOct 10, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 25, 2026Provider: repository / Type: Initial release
Revision 1.0Mar 25, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 25, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 25, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 25, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 25, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Mar 25, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
8: Penton protein
K: Penton protein
R: Penton protein
T: Penton protein
X: Penton protein
i: Penton protein
j: Penton protein
k: Penton protein
v: Penton protein
w: Penton protein


Theoretical massNumber of molelcules
Total (without water)608,54110
Polymers608,54110
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Penton protein / CP-P / Penton base protein / Protein III


Mass: 60854.137 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chimpanzee adenovirus Y25 / Gene: L2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: G9G849
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: CHIMPSELS S57C ADDomer: An engineered viral like particle derived from penton base protein of Chimpanzee adenovirus Y25.
Type: COMPLEX
Details: ADDomer produced by recombinant expression of the engineered penton base protein of Chimpanzee adenovirus Y25 using the MultiBac baculovirus-insect cell expression system. 60 penton base ...Details: ADDomer produced by recombinant expression of the engineered penton base protein of Chimpanzee adenovirus Y25 using the MultiBac baculovirus-insect cell expression system. 60 penton base proteins self assemble into the ADDomer particle.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 3.65 MDa / Experimental value: NO
Source (natural)Organism: Chimpanzee adenovirus Y25
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTrisTris1
2150 mMsodium chlorideNaCl1
30.2 mMEthylenediaminetetraacetic acidEDTA1
40.1 mM4-(2-Aminoethyl)benzenesulfonyl fluorideAEBSF1
5100 uMCopper PhenanthrolineDichloro(1,10-phenanthroline)copper(II)1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: glow discharged at 4 mA for 120 seconds / Grid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277.15 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: 2000 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 3.846 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 16511

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Processing

EM software
IDNameVersionCategoryDetails
1RELION4particle selectionRELION autopicking software was used
4GctfCTF correction
7UCSF Chimeramodel fitting
9RELION4initial Euler assignment
10RELION4final Euler assignment
11RELIONclassification
12RELION43D reconstruction
13PHENIX1.20.1_4487model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2464479
3D reconstructionResolution: 2.61 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 113117 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingDetails: The CHIMPSELS ADDomer model was used an initial model source.
Source name: Other / Type: experimental model

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