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- EMDB-6961: Cryo-EM structure of PCV2 VLP+Fab fragments of mAb-3H11 -

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Basic information

Entry
Database: EMDB / ID: EMD-6961
TitleCryo-EM structure of PCV2 VLP+Fab fragments of mAb-3H11
Map dataCryo-EM structure of complex of full length PCV2 VLP Fab fragments of mAb-3H11
Sample
  • Virus: Porcine circovirus 2
Function / homologyCircovirus capsid protein / Circovirus capsid superfamily / Circovirus capsid protein / viral capsid assembly / T=1 icosahedral viral capsid / viral penetration into host nucleus / symbiont entry into host cell / virion attachment to host cell / Capsid protein
Function and homology information
Biological speciesPorcine circovirus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 15.0 Å
AuthorsMo X / Yuan AY
CitationJournal: PLoS Pathog / Year: 2019
Title: Structural roles of PCV2 capsid protein N-terminus in PCV2 particle assembly and identification of PCV2 type-specific neutralizing epitope.
Authors: Xiaobing Mo / Xiangdong Li / Bo Yin / Junhua Deng / Kegong Tian / Adam Yuan /
Abstract: Postweaning multisystemic wasting disease (PMWS) in piglets caused by porcine circovirus type 2 (PCV2) is one of the major threats to most pig farms worldwide. Among all the PCV types, PCV2 is the ...Postweaning multisystemic wasting disease (PMWS) in piglets caused by porcine circovirus type 2 (PCV2) is one of the major threats to most pig farms worldwide. Among all the PCV types, PCV2 is the dominant genotype causing PMWS and associated diseases. Considerable efforts were made to study the virus-like-particle (VLP) assembly and the specific PCV2-associated epitope(s) in order to establish the solid foundation for engineered PCV2 vaccine development. Although the N-terminal fragment including Nuclear Localization Signal (NLS) sequence seems important for recombinant PCV2 capsid protein expression and VLP assembly, the detailed structural and functional information regarding this important fragment are largely unknown. In this study, we report crystal structure of PCV2 VLP assembled from N-terminal NLS truncated PCV2 capsid protein at 2.8 Å resolution and cryo-EM structure of PCV2 VLP assembled from full-length PCV2 capsid protein at 4.1Å resolution. Our in vitro PCV2 VLP assembly results show that NLS-truncated PCV2 capsid protein only forms instable VLPs which were easily disassembled in solution, whereas full-length PCV2 capsid protein forms stable VLPs due to interaction between 15PRSHLGQILRRRP27 (α-helix) and 33RHRYRWRRKN42 (NLS-B) in a repeated manner. In addition, our results also showed that N-terminal truncation of PCV2 capsid protein up to 27 residues still forms PCV2 particles in solution with similar size and immunogenicity, while N-terminal truncation of PCV2 capsid protein with more than 30 residues is not able to form stable PCV2 particles in solution, demonstrating the importance of interaction between the α-helix at N-terminal and NLS-B in PCV2 VLP formation. Moreover, we also report the cryo-EM structure of PCV2 VLP in complex with 3H11-Fab, a PCV2 type-specific neutralizing antibody, at 15 Å resolution. MAb-3H11 specifically recognizes one exposed epitope located on the VLP surface EF-loop (residues 128-143), which is further confirmed by PCV1-PCV2 epitope swapping assay. Hence, our results have revealed the structural roles of N-terminal fragment of PCV2 capsid protein in PCV2 particle assembly and pinpointed one PCV2 type-specific neutralizing epitope for the first time, which could provide clear clue for next generation PCV2 vaccine and diagnostic kits development.
History
DepositionMay 3, 2018-
Header (metadata) releaseMay 16, 2018-
Map releaseMay 16, 2018-
UpdateMar 13, 2019-
Current statusMar 13, 2019Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.34
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.34
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6961.png

Downloads & links

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Map

FileDownload / File: emd_6961.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of complex of full length PCV2 VLP Fab fragments of mAb-3H11
Voxel sizeX=Y=Z: 2.08 Å
Density
Contour LevelBy AUTHOR: 0.34 / Movie #1: 0.34
Minimum - Maximum-0.33655977 - 2.2297108
Average (Standard dev.)0.096851304 (±0.32684305)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-110-110-110
Dimensions220220220
Spacing220220220
CellA=B=C: 457.59998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.082.082.08
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z457.600457.600457.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-110-110-110
NC/NR/NS220220220
D min/max/mean-0.3372.2300.097

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Supplemental data

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Sample components

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Entire : Porcine circovirus 2

EntireName: Porcine circovirus 2
Components
  • Virus: Porcine circovirus 2

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Supramolecule #1: Porcine circovirus 2

SupramoleculeName: Porcine circovirus 2 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Porcine circovirus 2 capsid protein, and Fab fragents generated by papain cleavage of PCV2 type-specific antibody.
NCBI-ID: 85708 / Sci species name: Porcine circovirus 2 / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: Yes
Host systemOrganism: Escherichia coli-Pichia pastoris shuttle vector pPpARG4 (others)
Molecular weightExperimental: 4.7 MDa

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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

Concentration1 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
500.0 mMNaClSodium chloridesodium chloride
20.0 mMTrisTris

Details: 20mM Tris(pH 7.0), 500mM NaCl
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: FORMVAR / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 5 seconds before pluning.
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeFEI TITAN
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 3.0 µm / Calibrated defocus min: 1.0 µm / Calibrated magnification: 77271 / Illumination mode: OTHER / Imaging mode: DARK FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 47000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 70.0 K / Max: 70.0 K
DetailsPreliminary grid screening was performed manually.
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Number grids imaged: 5 / Number real images: 99 / Average exposure time: 1.0 sec. / Average electron dose: 35.0 e/Å2

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Image processing

Particle selectionNumber selected: 21280
CTF correctionSoftware - Name: EMAN2 (ver. 2.1)
Initial angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 32 / Avg.num./class: 200 / Software - Name: EMAN2 (ver. 2.1)
Final angle assignmentType: NOT APPLICABLE
Final reconstructionNumber classes used: 20 / Applied symmetry - Point group: I (icosahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN2 (ver. 2.1) / Number images used: 6500
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: AB INITIO MODEL / Overall B value: 200

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