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- EMDB-0838: Neutralization mechanism of a monoclonal antibody targeting a por... -

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Basic information

Entry
Database: EMDB / ID: EMD-0838
TitleNeutralization mechanism of a monoclonal antibody targeting a porcine circovirus type 2 Cap protein conformational epitope
Map data
Sample
  • Complex: PCV2 virion in complex with Fab fragments of the mAb 3A5
    • Complex: Light chain of Fab fragment of the mAb 3A5
      • Protein or peptide: Light chain of Fab fragment
    • Complex: Heavy chain of Fab fragment Fab of the mAb 3A5
      • Protein or peptide: Heavy chain of Fab fragment
    • Complex: PCV2 virion
      • Protein or peptide: Capsid protein
Function / homologyCircovirus capsid protein / Circovirus capsid superfamily / Circovirus capsid protein / viral capsid assembly / T=1 icosahedral viral capsid / symbiont entry into host cell / virion attachment to host cell / Capsid protein
Function and homology information
Biological speciesMus musculus (house mouse) / mus muscu (virus) / PCV2 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.2 Å
AuthorsSun Z / Huang L / Xia D / Wei Y / Sun E / Zhu H / Bian H / Wu H / Feng L / Wang J / Liu C
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31873012 China
CitationJournal: J Virol / Year: 2020
Title: Neutralization Mechanism of a Monoclonal Antibody Targeting a Porcine Circovirus Type 2 Cap Protein Conformational Epitope.
Authors: Liping Huang / Zhenzhao Sun / Deli Xia / Yanwu Wei / Encheng Sun / Chunguo Liu / Hongzhen Zhu / Haiqiao Bian / Hongli Wu / Li Feng / Jingfei Wang / Changming Liu /
Abstract: Porcine circovirus type 2 (PCV2) is an important pathogen in swine herds, and its infection of pigs has caused severe economic losses to the pig industry worldwide. The capsid protein of PCV2 is the ...Porcine circovirus type 2 (PCV2) is an important pathogen in swine herds, and its infection of pigs has caused severe economic losses to the pig industry worldwide. The capsid protein of PCV2 is the only structural protein that is associated with PCV2 infection and immunity. Here, we report a neutralizing monoclonal antibody (MAb), MAb 3A5, that binds to intact PCV2 virions of the PCV2a, PCV2b, and PCV2d genotypes. MAb 3A5 neutralized PCV2 by blocking viral attachment to PK15 cells. To further explore the neutralization mechanism, we resolved the structure of the PCV2 virion in complex with MAb 3A5 Fab fragments by using cryo-electron microscopy single-particle analysis. The binding sites were located at the topmost edges around 5-fold icosahedral symmetry axes, with each footprint covering amino acids from two adjacent capsid proteins. Most of the epitope residues (15/18 residues) were conserved among 2,273 PCV2 strains. Mutations of some amino acids within the epitope had significant effects on the neutralizing activity of MAb 3A5. This study reveals the molecular and structural bases of this PCV2-neutralizing antibody and provides new and important information for vaccine design and therapeutic antibody development against PCV2 infections. PCV2 is associated with several clinical manifestations collectively known as PCV2-associated diseases (PCVADs). Neutralizing antibodies play a crucial role in the prevention of PCVADs. We demonstrated previously that a MAb, MAb 3A5, neutralizes the PCV2a, PCV2b, and PCV2d genotypes with different degrees of efficiency, but the underlying mechanism remains elusive. Here, we report the neutralization mechanism of this MAb and the structure of the PCV2 virion in complex with MAb 3A5 Fabs, showing a binding mode in which one Fab interacted with more than two loops from two adjacent capsid proteins. This binding mode has not been observed previously for PCV2-neutralizing antibodies. Our work provides new and important information for vaccine design and therapeutic antibody development against PCV2 infections.
History
DepositionOct 25, 2019-
Header (metadata) releaseFeb 12, 2020-
Map releaseFeb 12, 2020-
UpdateApr 29, 2020-
Current statusApr 29, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
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  • Surface view with fitted model
  • Atomic models: PDB-6l62
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6l62
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0838.png

Downloads & links

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Map

FileDownload / File: emd_0838.map.gz / Format: CCP4 / Size: 347.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.14 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.028893631 - 0.0794303
Average (Standard dev.)0.0012859281 (±0.006237077)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-225-225-225
Dimensions450450450
Spacing450450450
CellA=B=C: 513.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.141.141.14
M x/y/z450450450
origin x/y/z0.0000.0000.000
length x/y/z513.000513.000513.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-225-225-225
NC/NR/NS450450450
D min/max/mean-0.0290.0790.001

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Supplemental data

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Sample components

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Entire : PCV2 virion in complex with Fab fragments of the mAb 3A5

EntireName: PCV2 virion in complex with Fab fragments of the mAb 3A5
Components
  • Complex: PCV2 virion in complex with Fab fragments of the mAb 3A5
    • Complex: Light chain of Fab fragment of the mAb 3A5
      • Protein or peptide: Light chain of Fab fragment
    • Complex: Heavy chain of Fab fragment Fab of the mAb 3A5
      • Protein or peptide: Heavy chain of Fab fragment
    • Complex: PCV2 virion
      • Protein or peptide: Capsid protein

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Supramolecule #1: PCV2 virion in complex with Fab fragments of the mAb 3A5

SupramoleculeName: PCV2 virion in complex with Fab fragments of the mAb 3A5
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #2: Light chain of Fab fragment of the mAb 3A5

SupramoleculeName: Light chain of Fab fragment of the mAb 3A5 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #3: Heavy chain of Fab fragment Fab of the mAb 3A5

SupramoleculeName: Heavy chain of Fab fragment Fab of the mAb 3A5 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: mus muscu (virus)

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Supramolecule #4: PCV2 virion

SupramoleculeName: PCV2 virion / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3

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Macromolecule #1: Light chain of Fab fragment

MacromoleculeName: Light chain of Fab fragment / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.564074 KDa
SequenceString: NIVMTQSPKS MSMSVGERVT LSCKASENVG TFVFWYQQKP EQSPQLLIYG ASNRYTGVPD RFTGSGSATD FTLTINNVQA EDFVDYYCG QSYRYPLTFA AGTKLGLKRA DAAPTVSIFP PSSEQLTSGG ASVVCFLNNF YPKDINVKWK IDGSERQNGV L NSWTDQDS ...String:
NIVMTQSPKS MSMSVGERVT LSCKASENVG TFVFWYQQKP EQSPQLLIYG ASNRYTGVPD RFTGSGSATD FTLTINNVQA EDFVDYYCG QSYRYPLTFA AGTKLGLKRA DAAPTVSIFP PSSEQLTSGG ASVVCFLNNF YPKDINVKWK IDGSERQNGV L NSWTDQDS KDSTYSMSST LTLTKDEYER HNSYTCEATH KTSTSPIVKS FNRN

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Macromolecule #2: Heavy chain of Fab fragment

MacromoleculeName: Heavy chain of Fab fragment / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.728473 KDa
SequenceString: QVQLQQSGAE LVRPGVSVKI SCKGSGYTFT DYAIHWVKQS HAKSLEWIGL ISTYYGDATY NQNFKGEATM TVDKSSSTAY MELARLTSE DSAIYYCARG PFSRYDYFAM DNWGQGTSVT VSSAKTTPPS VYPLAPGSAA QTNSMVTLGC LVKGYFPEPV T VTWNSGSL ...String:
QVQLQQSGAE LVRPGVSVKI SCKGSGYTFT DYAIHWVKQS HAKSLEWIGL ISTYYGDATY NQNFKGEATM TVDKSSSTAY MELARLTSE DSAIYYCARG PFSRYDYFAM DNWGQGTSVT VSSAKTTPPS VYPLAPGSAA QTNSMVTLGC LVKGYFPEPV T VTWNSGSL SSGVHTFPAV LQSDLYTLSS SVTVPSSTWP SETVTCNVAH PASSTKVDKK IV

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Macromolecule #3: Capsid protein

MacromoleculeName: Capsid protein / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: PCV2 (virus)
Molecular weightTheoretical: 27.55952 KDa
SequenceString: MTYPRRRFRR RRHRPRSHLG LILRRRPWLV HPRHRYRWRR KNGIFNTRLS CTFGYTVKAT TVRTPSWAVD MMRFNINDFV PPGGGTNKI SIPFEYYRIR KVKVEFWPCS PITQGDRGVG STAVILDDNF VTKATALTYD PYVNYSSRHT IPQPFSYHSR Y FTPKPVLD ...String:
MTYPRRRFRR RRHRPRSHLG LILRRRPWLV HPRHRYRWRR KNGIFNTRLS CTFGYTVKAT TVRTPSWAVD MMRFNINDFV PPGGGTNKI SIPFEYYRIR KVKVEFWPCS PITQGDRGVG STAVILDDNF VTKATALTYD PYVNYSSRHT IPQPFSYHSR Y FTPKPVLD STIDYFQPNN KRNQLWLRLQ TSANVDHVGL GIAFENSTYD QDYNIRVTMY VQFREFNLKD PPL

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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: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recording#0 - Image recording ID: 1 / #0 - Film or detector model: FEI CETA (4k x 4k) / #0 - Average electron dose: 35.0 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: FEI CETA (4k x 4k) / #1 - Average electron dose: 35.0 e/Å2 / #2 - Image recording ID: 3 / #2 - Film or detector model: FEI CETA (4k x 4k) / #2 - Average electron dose: 35.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Image recording ID1
Final reconstructionResolution.type: BY AUTHOR / Resolution: 7.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 3500
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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