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- EMDB-28807: Cryo-EM structure of Lumazine synthase nanoparticle linked to VP8... -

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Basic information

Entry
Database: EMDB / ID: EMD-28807
TitleCryo-EM structure of Lumazine synthase nanoparticle linked to VP8* antigen
Map dataCryo-EM structure of Lumazine synthase nanoparticle linked to VP8* antigen
Sample
  • Complex: Lumazine synthase nanoparticle linked to rotatvirus VP8* protein
    • Protein or peptide: 6,7-dimethyl-8-ribityllumazine synthase
KeywordsRotavirus / VP8* / lumazine synthase / nanoparticle / VIRAL PROTEIN
Function / homology
Function and homology information


6,7-dimethyl-8-ribityllumazine synthase / 6,7-dimethyl-8-ribityllumazine synthase activity / riboflavin synthase complex / riboflavin biosynthetic process / cytoplasm / cytosol
Similarity search - Function
Lumazine synthase / Lumazine/riboflavin synthase / Lumazine/riboflavin synthase superfamily / 6,7-dimethyl-8-ribityllumazine synthase
Similarity search - Domain/homology
6,7-dimethyl-8-ribityllumazine synthase
Similarity search - Component
Biological speciesAquifex aeolicus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsMangala Prasad V / Lee KK
Funding support United States, 1 items
OrganizationGrant numberCountry
Bill & Melinda Gates Foundation United States
CitationJournal: NPJ Vaccines / Year: 2023
Title: mRNA-based VP8* nanoparticle vaccines against rotavirus are highly immunogenic in rodents.
Authors: Sandro Roier / Vidya Mangala Prasad / Monica M McNeal / Kelly K Lee / Benjamin Petsch / Susanne Rauch /
Abstract: Despite the availability of live-attenuated oral vaccines, rotavirus remains a major cause of severe childhood diarrhea worldwide. Due to the growing demand for parenteral rotavirus vaccines, we ...Despite the availability of live-attenuated oral vaccines, rotavirus remains a major cause of severe childhood diarrhea worldwide. Due to the growing demand for parenteral rotavirus vaccines, we developed mRNA-based vaccine candidates targeting the viral spike protein VP8*. Our monomeric P2 (universal T cell epitope)-VP8* mRNA design is equivalent to a protein vaccine currently in clinical development, while LS (lumazine synthase)-P2-VP8* was designed to form nanoparticles. Cyro-electron microscopy and western blotting-based data presented here suggest that proteins derived from LS-P2-VP8* mRNA are secreted in vitro and self-assemble into 60-mer nanoparticles displaying VP8*. mRNA encoded VP8* was immunogenic in rodents and introduced both humoral and cellular responses. LS-P2-VP8* induced superior humoral responses to P2-VP8* in guinea pigs, both as monovalent and trivalent vaccines, with encouraging responses detected against the most prevalent P genotypes. Overall, our data provide evidence that trivalent LS-P2-VP8* represents a promising mRNA-based next-generation rotavirus vaccine candidate.
History
DepositionNov 7, 2022-
Header (metadata) releaseApr 12, 2023-
Map releaseApr 12, 2023-
UpdateJan 10, 2024-
Current statusJan 10, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28807.png

Downloads & links

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Map

FileDownload / File: emd_28807.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of Lumazine synthase nanoparticle linked to VP8* antigen
Projections & slices

Image control

Size
Brightness
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Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.06 Å/pix.
x 288 pix.
= 305.28 Å		1.06 Å/pix.
x 288 pix.
= 305.28 Å		1.06 Å/pix.
x 288 pix.
= 305.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.0
Minimum - Maximum-6.2372518 - 17.690763
Average (Standard dev.)0.000000000003827 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 305.27997 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half-map2

Fileemd_28807_half_map_1.map
AnnotationHalf-map2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map

Fileemd_28807_half_map_2.map
AnnotationHalf-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 : Lumazine synthase nanoparticle linked to rotatvirus VP8* protein

EntireName: Lumazine synthase nanoparticle linked to rotatvirus VP8* protein
Components
  • Complex: Lumazine synthase nanoparticle linked to rotatvirus VP8* protein
    • Protein or peptide: 6,7-dimethyl-8-ribityllumazine synthase

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Supramolecule #1: Lumazine synthase nanoparticle linked to rotatvirus VP8* protein

SupramoleculeName: Lumazine synthase nanoparticle linked to rotatvirus VP8* protein
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Aquifex aeolicus (bacteria)
Molecular weightTheoretical: 2.7 MDa

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Macromolecule #1: 6,7-dimethyl-8-ribityllumazine synthase

MacromoleculeName: 6,7-dimethyl-8-ribityllumazine synthase / type: protein_or_peptide / ID: 1 / Number of copies: 60 / Enantiomer: LEVO / EC number: 6,7-dimethyl-8-ribityllumazine synthase
Source (natural)Organism: Aquifex aeolicus (bacteria) / Strain: VF5
Molecular weightTheoretical: 16.728186 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
MEIYEGKLTA EGLRFGIVAS RFNHALVDRL VEGAIDCIVR HGGREEDITL VRVPGSWEIP VAAGELARKE DIDAVIAIGV LIRGATPHF DYIASEVSKG LANLSLELRK PITFGVITAD TLEQAIERAG TKHGNKGWEA ALSAIEMANL FKSLR

UniProtKB: 6,7-dimethyl-8-ribityllumazine synthase

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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

Concentration0.5 mg/mL
BufferpH: 7.4 / Details: Phosphate Buffer Saline
GridModel: EMS Lacey Carbon / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Detailsmonodisperse sample in PBS

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average exposure time: 10.0 sec. / Average electron dose: 284.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.75 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 135718 / Details: Automated particle picking
Startup modelType of model: EMDB MAP
EMDB ID:

3538

Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1936
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsRigid body fitting followed by default Real space refinement in phenix with only rigid body fitting and occupancy refinement
RefinementProtocol: RIGID BODY FIT
Output model

PDB-8f25:
Cryo-EM structure of Lumazine synthase nanoparticle linked to VP8* antigen

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