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- EMDB-27812: Structure of engineered nano-cage fusion protein -

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Basic information

Entry
Database: EMDB / ID: EMD-27812
TitleStructure of engineered nano-cage fusion protein
Map datasharpened icosahedral map
Sample
  • Complex: Nano-cage
    • Protein or peptide: 2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsMoustafa IM / Hafenstein SL
Funding support United States, 1 items
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, 2022-
Header (metadata) releaseNov 16, 2022-
Map releaseNov 16, 2022-
UpdateDec 28, 2022-
Current statusDec 28, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27812.png

Downloads & links

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Map

FileDownload / File: emd_27812.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened icosahedral map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 420 pix.
= 462. Å		1.1 Å/pix.
x 420 pix.
= 462. Å		1.1 Å/pix.
x 420 pix.
= 462. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.9
Minimum - Maximum-4.024448 - 6.3624315
Average (Standard dev.)0.0073766955 (±0.21626735)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-209-209-209
Dimensions420420420
Spacing420420420
CellA=B=C: 462.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_27812_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfB map

Fileemd_27812_half_map_1.map
AnnotationhalfB map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: halfA map

Fileemd_27812_half_map_2.map
AnnotationhalfA map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Nano-cage

EntireName: Nano-cage
Components
  • Complex: Nano-cage
    • Protein or peptide: 2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase

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Supramolecule #1: Nano-cage

SupramoleculeName: Nano-cage / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Thermotoga maritima (bacteria)
Molecular weightTheoretical: 1.3 MDa

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Macromolecule #1: 2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutara...

MacromoleculeName: 2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermotoga maritima (bacteria)
Molecular weightTheoretical: 23.915986 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MHHHHHHGGS GGSGGSGGSM KMEELFKKHK IVAVLRANSV EEAKKKALAV FLGGVHLIEI TFTVPDADTV IKELSFLKEM GAIIGAGTV TSVEQCRKAV ESGAEFIVSP HLDEEISQFC KEKGVFYMPG VMTPTELVKA MKLGHTILKL FPGEVVGPQF V KAMKGPFP ...String:
MHHHHHHGGS GGSGGSGGSM KMEELFKKHK IVAVLRANSV EEAKKKALAV FLGGVHLIEI TFTVPDADTV IKELSFLKEM GAIIGAGTV TSVEQCRKAV ESGAEFIVSP HLDEEISQFC KEKGVFYMPG VMTPTELVKA MKLGHTILKL FPGEVVGPQF V KAMKGPFP NVKFVPTGGV NLDNVCEWFK AGVLAVGVGS ALVKGTPVEV AEKAKAFVEK IRGCTE

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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

BufferpH: 8.1 / Details: Tris, 100 mM NaCl, 1 mM DTT
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsPurified nano-cage protein at 0.1 mg/mL

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 100.0 K
Specialist opticsSpherical aberration corrector: Microscope was modified with a Cs corrector.
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 1220 / Average exposure time: 69.8 sec. / Average electron dose: 44.85 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 0.05 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 59000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 129297
Startup modelType of model: INSILICO MODEL / In silico model: I3-01
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 63430
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3.3.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 69.1 / Target criteria: Correlation coefficient
Output model

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

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