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

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Basic information

Entry
Database: PDB / ID: 8f25
TitleCryo-EM structure of Lumazine synthase nanoparticle linked to VP8* antigen
Components6,7-dimethyl-8-ribityllumazine synthase
KeywordsVIRAL PROTEIN / Rotavirus / VP8* / lumazine synthase / nanoparticle
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 / cytosol / cytoplasm
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsMangala Prasad, V. / Lee, K.K.
Funding support United States, 1items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 12, 2023Provider: repository / Type: Initial release
Revision 1.1Jan 10, 2024Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: 6,7-dimethyl-8-ribityllumazine synthase
B: 6,7-dimethyl-8-ribityllumazine synthase
C: 6,7-dimethyl-8-ribityllumazine synthase
D: 6,7-dimethyl-8-ribityllumazine synthase
E: 6,7-dimethyl-8-ribityllumazine synthase
F: 6,7-dimethyl-8-ribityllumazine synthase
G: 6,7-dimethyl-8-ribityllumazine synthase
H: 6,7-dimethyl-8-ribityllumazine synthase
I: 6,7-dimethyl-8-ribityllumazine synthase
J: 6,7-dimethyl-8-ribityllumazine synthase
K: 6,7-dimethyl-8-ribityllumazine synthase
L: 6,7-dimethyl-8-ribityllumazine synthase
M: 6,7-dimethyl-8-ribityllumazine synthase
N: 6,7-dimethyl-8-ribityllumazine synthase
O: 6,7-dimethyl-8-ribityllumazine synthase
P: 6,7-dimethyl-8-ribityllumazine synthase
Q: 6,7-dimethyl-8-ribityllumazine synthase
R: 6,7-dimethyl-8-ribityllumazine synthase
S: 6,7-dimethyl-8-ribityllumazine synthase
T: 6,7-dimethyl-8-ribityllumazine synthase
U: 6,7-dimethyl-8-ribityllumazine synthase
V: 6,7-dimethyl-8-ribityllumazine synthase
W: 6,7-dimethyl-8-ribityllumazine synthase
X: 6,7-dimethyl-8-ribityllumazine synthase
Y: 6,7-dimethyl-8-ribityllumazine synthase
Z: 6,7-dimethyl-8-ribityllumazine synthase
0: 6,7-dimethyl-8-ribityllumazine synthase
1: 6,7-dimethyl-8-ribityllumazine synthase
2: 6,7-dimethyl-8-ribityllumazine synthase
3: 6,7-dimethyl-8-ribityllumazine synthase
4: 6,7-dimethyl-8-ribityllumazine synthase
5: 6,7-dimethyl-8-ribityllumazine synthase
6: 6,7-dimethyl-8-ribityllumazine synthase
7: 6,7-dimethyl-8-ribityllumazine synthase
8: 6,7-dimethyl-8-ribityllumazine synthase
9: 6,7-dimethyl-8-ribityllumazine synthase
a: 6,7-dimethyl-8-ribityllumazine synthase
b: 6,7-dimethyl-8-ribityllumazine synthase
c: 6,7-dimethyl-8-ribityllumazine synthase
d: 6,7-dimethyl-8-ribityllumazine synthase
e: 6,7-dimethyl-8-ribityllumazine synthase
f: 6,7-dimethyl-8-ribityllumazine synthase
g: 6,7-dimethyl-8-ribityllumazine synthase
h: 6,7-dimethyl-8-ribityllumazine synthase
i: 6,7-dimethyl-8-ribityllumazine synthase
j: 6,7-dimethyl-8-ribityllumazine synthase
k: 6,7-dimethyl-8-ribityllumazine synthase
l: 6,7-dimethyl-8-ribityllumazine synthase
m: 6,7-dimethyl-8-ribityllumazine synthase
n: 6,7-dimethyl-8-ribityllumazine synthase
o: 6,7-dimethyl-8-ribityllumazine synthase
p: 6,7-dimethyl-8-ribityllumazine synthase
q: 6,7-dimethyl-8-ribityllumazine synthase
r: 6,7-dimethyl-8-ribityllumazine synthase
s: 6,7-dimethyl-8-ribityllumazine synthase
t: 6,7-dimethyl-8-ribityllumazine synthase
u: 6,7-dimethyl-8-ribityllumazine synthase
v: 6,7-dimethyl-8-ribityllumazine synthase
w: 6,7-dimethyl-8-ribityllumazine synthase
x: 6,7-dimethyl-8-ribityllumazine synthase


Theoretical massNumber of molelcules
Total (without water)1,003,69160
Polymers1,003,69160
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ...
6,7-dimethyl-8-ribityllumazine synthase / DMRL synthase / LS / Lumazine synthase


Mass: 16728.186 Da / Num. of mol.: 60
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aquifex aeolicus (bacteria) / Strain: VF5 / Gene: ribH, aq_132 / Production host: Homo sapiens (human)
References: UniProt: O66529, 6,7-dimethyl-8-ribityllumazine synthase

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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: Lumazine synthase nanoparticle linked to rotatvirus VP8* protein
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 2.7 MDa / Experimental value: NO
Source (natural)Organism: Aquifex aeolicus (bacteria)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK
Buffer solutionpH: 7.4 / Details: Phosphate Buffer Saline
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: monodisperse sample in PBS
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: EMS Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 750 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 284 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameCategory
1RELIONparticle selection
2Leginonimage acquisition
4CTFFINDCTF correction
7UCSF Chimeramodel fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 135718 / Details: Automated particle picking
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1936 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Details: Rigid body fitting followed by default Real space refinement in phenix with only rigid body fitting and occupancy refinement
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00771640
ELECTRON MICROSCOPYf_angle_d0.68596780
ELECTRON MICROSCOPYf_dihedral_angle_d4.4449960
ELECTRON MICROSCOPYf_chiral_restr0.04911220
ELECTRON MICROSCOPYf_plane_restr0.00512480

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