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- PDB-8e01: Structure of engineered nano-cage fusion protein -

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Basic information

Entry
Database: PDB / ID: 80
TitleStructure of engineered nano-cage fusion protein
Components2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase
KeywordsPROTEIN BINDING / nano-cage / self-assembly / fusion protein
Function / homologyKDPG/KHG aldolase / KDPG and KHG aldolase / Aldolase-type TIM barrel / lyase activity / 2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase
Function and homology information
Biological speciesThermotoga maritima (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsMoustafa, I.M. / Hafenstein, S.L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM125907 United States
CitationJournal: bioRxiv / Year: 2022
Title: Intranasal SARS-CoV-2 RBD decorated nanoparticle vaccine enhances viral clearance in the Syrian hamster model.
Authors: D R Patel / A M Minns / D G Sim / C J Field / A E Kerr / T Heinly / E H Luley / R M Rossi / C Bator / I M Moustafa / S L Hafenstein / S E Lindner / T C Sutton /
Abstract: Multiple vaccines have been developed and licensed for SARS-CoV-2. While these vaccines reduce disease severity, they do not prevent infection, and SARS-CoV-2 continues to spread and evolve. To ...Multiple vaccines have been developed and licensed for SARS-CoV-2. While these vaccines reduce disease severity, they do not prevent infection, and SARS-CoV-2 continues to spread and evolve. To prevent infection and limit transmission, vaccines must be developed that induce immunity in the respiratory tract. Therefore, we performed proof-of-principle vaccination studies with an intranasal nanoparticle vaccine against SARS-CoV-2. The vaccine candidate consisted of the self-assembling 60-subunit I3-01 protein scaffold covalently decorated with the SARS-CoV-2 receptor binding domain (RBD) using the SpyCatcher-SpyTag system. We verified the intended antigen display features by reconstructing the I3-01 scaffold to 3.4A using cryo-EM, and then demonstrated that the scaffold was highly saturated when grafted with RBD. Using this RBD-grafted SpyCage scaffold (RBD+SpyCage), we performed two unadjuvanted intranasal vaccination studies in the "gold-standard" preclinical Syrian hamster model. Hamsters received two vaccinations 28 days apart, and were then challenged 28 days post-boost with SARS-CoV-2. The initial study focused on assessing the immunogenicity of RBD+SpyCage, which indicated that vaccination of hamsters induced a non-neutralizing antibody response that enhanced viral clearance but did not prevent infection. In an expanded study, we demonstrated that covalent bonding of RBD to the scaffold was required to induce an antibody response. Consistent with the initial study, animals vaccinated with RBD+SpyCage more rapidly cleared SARS-CoV-2 from both the upper and lower respiratory tract. These findings demonstrate the intranasal SpyCage vaccine platform can induce protection against SARS-CoV-2 and, with additional modifications to improve immunogenicity, is a versatile platform for the development of intranasal vaccines targeting respiratory pathogens.
History
DepositionAug 8, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 16, 2022Provider: repository / Type: Initial release
Revision 1.1Dec 28, 2022Group: Database references / Category: citation / citation_author / Item: _citation.title / _citation_author.name
Revision 1.2Nov 20, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: 2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase


Theoretical massNumber of molelcules
Total (without water)23,9161
Polymers23,9161
Non-polymers00
Water00
1
A: 2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase
x 60


Theoretical massNumber of molelcules
Total (without water)1,434,95960
Polymers1,434,95960
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59
2


  • Idetical with deposited unit
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
A: 2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase
x 5


  • icosahedral pentamer
  • 120 kDa, 5 polymers
Theoretical massNumber of molelcules
Total (without water)119,5805
Polymers119,5805
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: 2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase
x 6


  • icosahedral 23 hexamer
  • 143 kDa, 6 polymers
Theoretical massNumber of molelcules
Total (without water)143,4966
Polymers143,4966
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation5
5


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Protein 2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase / Engineered nano-cage fusion protein


Mass: 23915.986 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermotoga maritima (bacteria) / Gene: TM_0066 / Plasmid: pSL1013 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: Q9WXS1
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: Nano-cage / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.3 MDa / Experimental value: NO
Source (natural)Organism: Thermotoga maritima (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8.1 / Details: Tris, 100 mM NaCl, 1 mM DTT
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Purified nano-cage protein at 0.1 mg/mL
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 59000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1200 nm / Cs: 0.05 mm
Specimen holderCryogen: NITROGEN / Temperature (min): 100 K
Image recordingAverage exposure time: 69.8 sec. / Electron dose: 44.85 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1220
EM imaging opticsSpherical aberration corrector: Microscope was modified with a Cs corrector.
Image scansWidth: 4096 / Height: 4096

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Processing

SoftwareName: PHENIX / Version: 1.20.1-4487_4487: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARC3.3.2particle selection
2EPUimage acquisitionThermo Ficher specialized software
4cryoSPARC3.3.2CTF correction
7UCSF ChimeraX1.3model fitting
9cryoSPARC3.3.2initial Euler assignment
10cryoSPARC3.3.2final Euler assignment
11cryoSPARC3.3.2classification
12cryoSPARC3.3.23D reconstruction
13PHENIX1.20.1-4487-4487model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 129297
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 63430 / Symmetry type: POINT
Atomic model buildingB value: 69.1 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation coefficient
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00292880
ELECTRON MICROSCOPYf_angle_d0.514125280
ELECTRON MICROSCOPYf_dihedral_angle_d5.1512300
ELECTRON MICROSCOPYf_chiral_restr0.04314700
ELECTRON MICROSCOPYf_plane_restr0.00415780

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