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- PDB-7uja: Cryo-EM structure of Human respiratory syncytial virus F variant ... -

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Basic information

Entry
Database: PDB / ID: 7uja
TitleCryo-EM structure of Human respiratory syncytial virus F variant (construct pXCS847A)
Components
  • (RSV variant (construct pXCS847A) ...) x 2
  • AM14 Fab heavy chain
  • AM14 Fab light chain
  • AM22 Fab heavy chain
  • AM22 Fab light chain
KeywordsVIRAL PROTEIN / RSV / RSVF / pre-fusion
Function / homologyPrecursor fusion glycoprotein F0, Paramyxoviridae / Fusion glycoprotein F0 / fusion of virus membrane with host plasma membrane / host cell plasma membrane / virion membrane / plasma membrane / Fusion glycoprotein F0
Function and homology information
Biological speciesRespiratory syncytial virus
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsLees, J.A. / Ammirati, M. / Han, S.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Sci Transl Med / Year: 2023
Title: Rational design of a highly immunogenic prefusion-stabilized F glycoprotein antigen for a respiratory syncytial virus vaccine.
Authors: Ye Che / Alexey V Gribenko / Xi Song / Luke D Handke / Kari S Efferen / Kristin Tompkins / Srinivas Kodali / Lorna Nunez / A Krishna Prasad / Lynn M Phelan / Mark Ammirati / Xiaodi Yu / ...Authors: Ye Che / Alexey V Gribenko / Xi Song / Luke D Handke / Kari S Efferen / Kristin Tompkins / Srinivas Kodali / Lorna Nunez / A Krishna Prasad / Lynn M Phelan / Mark Ammirati / Xiaodi Yu / Joshua A Lees / Wei Chen / Lyndsey Martinez / Vidia Roopchand / Seungil Han / Xiayang Qiu / John P DeVincenzo / Kathrin U Jansen / Philip R Dormitzer / Kena A Swanson /
Abstract: Respiratory syncytial virus (RSV) is the leading, global cause of serious respiratory disease in infants and is an important cause of respiratory illness in older adults. No RSV vaccine is currently ...Respiratory syncytial virus (RSV) is the leading, global cause of serious respiratory disease in infants and is an important cause of respiratory illness in older adults. No RSV vaccine is currently available. The RSV fusion (F) glycoprotein is a key antigen for vaccine development, and its prefusion conformation is the target of the most potent neutralizing antibodies. Here, we describe a computational and experimental strategy for designing immunogens that enhance the conformational stability and immunogenicity of RSV prefusion F. We obtained an optimized vaccine antigen after screening nearly 400 engineered F constructs. Through in vitro and in vivo characterization studies, we identified F constructs that are more stable in the prefusion conformation and elicit ~10-fold higher serum-neutralizing titers in cotton rats than DS-Cav1. The stabilizing mutations of the lead construct (847) were introduced onto F glycoprotein backbones of strains representing the dominant circulating genotypes of the two major RSV subgroups, A and B. Immunization of cotton rats with a bivalent vaccine formulation of these antigens conferred complete protection against RSV challenge, with no evidence of disease enhancement. The resulting bivalent RSV prefusion F investigational vaccine has recently been shown to be efficacious against RSV disease in two pivotal phase 3 efficacy trials, one for passive protection of infants by immunization of pregnant women and the second for active protection of older adults by direct immunization.
History
DepositionMar 30, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 19, 2023Provider: repository / Type: Initial release
Revision 1.1May 10, 2023Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RSV variant (construct pXCS847A) F1
B: AM14 Fab heavy chain
C: RSV variant (construct pXCS847A) F1
D: AM14 Fab light chain
E: AM22 Fab heavy chain
F: AM22 Fab light chain
G: AM14 Fab heavy chain
I: AM22 Fab heavy chain
J: AM22 Fab light chain
K: RSV variant (construct pXCS847A) F1
L: AM14 Fab heavy chain
N: AM22 Fab heavy chain
O: AM22 Fab light chain
H: AM14 Fab light chain
M: AM14 Fab light chain
P: RSV variant (construct pXCS847A) F2
Q: RSV variant (construct pXCS847A) F2
R: RSV variant (construct pXCS847A) F2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)470,47021
Polymers469,80718
Non-polymers6643
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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RSV variant (construct pXCS847A) ... , 2 types, 6 molecules ACKPQR

#1: Protein RSV variant (construct pXCS847A) F1


Mass: 45025.391 Da / Num. of mol.: 3 / Mutation: I145S, S187I, D483S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Respiratory syncytial virus / Production host: Mammalia (mammals) / References: UniProt: A0A7D5GVC1
#6: Protein RSV variant (construct pXCS847A) F2


Mass: 12109.954 Da / Num. of mol.: 3 / Mutation: A100C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Respiratory syncytial virus / Production host: Mammalia (mammals) / References: UniProt: A0A7D5GVC1

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Antibody , 4 types, 12 molecules BGLDHMEINFJO

#2: Antibody AM14 Fab heavy chain


Mass: 26231.088 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mammalia (mammals)
#3: Antibody AM14 Fab light chain


Mass: 23972.656 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mammalia (mammals)
#4: Antibody AM22 Fab heavy chain


Mass: 25989.154 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mammalia (mammals)
#5: Antibody AM22 Fab light chain


Mass: 23273.938 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mammalia (mammals)

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Sugars , 1 types, 3 molecules

#7: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Details

Has ligand of interestN

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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: Human respiratory syncytial virus F variant (construct pXCS847A) with Fabs AM14 and AM22
Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Source (natural)Organism: Respiratory syncytial virus
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris HydrochlorideTris-HClTris1
2300 mMSodium chlorideNaClSodium chloride1
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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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 FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 5000 nm / Nominal defocus min: 500 nm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 20 sec. / Electron dose: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20_4459: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4CTF correction
9RELION3initial Euler assignment
10RELION3final Euler assignment
12RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 420325
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 183713 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00221126
ELECTRON MICROSCOPYf_angle_d0.54328647
ELECTRON MICROSCOPYf_dihedral_angle_d3.9132898
ELECTRON MICROSCOPYf_chiral_restr0.0423330
ELECTRON MICROSCOPYf_plane_restr0.0043630

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