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- PDB-3j93: Fitting of Fab into the cryoEM density map of EV71 procapsid in c... -

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Entry
Database: PDB / ID: 3j93
TitleFitting of Fab into the cryoEM density map of EV71 procapsid in complex with Fab22A12
Components
  • neutralizing antibody 22A12, heavy chain
  • neutralizing antibody 22A12, light chain
KeywordsIMMUNE SYSTEM / Fab / EV71 / Fab22A12
Specimen sourceMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 8.8 Å resolution
AuthorsShingler, K.L. / Cifuente, J.O. / Ashley, R.E. / Makhov, A.M. / Conway, J.F. / Hafenstein, S.
CitationJournal: J. Virol. / Year: 2015
Title: The enterovirus 71 procapsid binds neutralizing antibodies and rescues virus infection in vitro.
Authors: Kristin L Shingler / Javier O Cifuente / Robert E Ashley / Alexander M Makhov / James F Conway / Susan Hafenstein
Abstract: Enterovirus 71 (EV71) is responsible for seasonal outbreaks of hand, foot, and mouth disease in the Asia-Pacific region. The virus has the capability to cause severe disease and death, especially in ...Enterovirus 71 (EV71) is responsible for seasonal outbreaks of hand, foot, and mouth disease in the Asia-Pacific region. The virus has the capability to cause severe disease and death, especially in young children. Although several vaccines are currently in clinical trials, no vaccines or therapeutics have been approved for use. Previous structural studies have revealed that two antigenically distinct capsid forms are produced in EV71-infected cells: an expanded empty capsid, sometimes called a procapsid, and the infectious virus. Specifically, an immunodominant epitope of EV71 that maps to the virus canyon is structurally different in the procapsid and virus. This structure-function study shows that the procapsid can sequester antibodies, thus enhancing EV71 infection in vitro. The results presented here suggest that, due to conformational differences between the EV71 procapsid and virus, the presence of the procapsid in natural virus infections should be considered in the future design of vaccines or therapeutics.
IMPORTANCE: In a picornavirus infection, both an infectious and a noninfectious empty capsid, sometimes referred to as a procapsid, are produced. It was novel to discover that the procapsid form of EV71 was expanded and antigenically distinct from the infectious virus. Previously, it had been supposed that this empty capsid was an off-pathway dead end or at best served for storage of pentameric subunits, which was later shown to be unlikely. It remains unexplained why picornaviruses evolutionarily conserve the wasteful production of so much noninfectious capsid. Here, we demonstrate that the EV71 procapsid has different antigenic properties than the infectious virus. Thus, the procapsid has the capacity to sequester neutralizing antibody and protect the virus, promoting or restoring a successful infection in vitro. This important observation should be considered in the future design and development of vaccines and therapeutics.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Dec 2, 2014 / Release: Dec 24, 2014
RevisionDateData content typeGroupCategoryItemProviderType
1.0Dec 24, 2014Structure modelrepositoryInitial release
1.1Jan 21, 2015Structure modelOther
1.2Jan 28, 2015Structure modelDatabase references
1.3Jul 18, 2018Structure modelData collectionem_image_scans / em_software_em_software.image_processing_id / _em_software.name

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Structure visualization

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  • Biological unit as complete icosahedral assembly
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  • Biological unit as icosahedral pentamer
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  • Biological unit as icosahedral 23 hexamer
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
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Structure viewerMolecule:
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Assembly

Deposited unit
L: neutralizing antibody 22A12, light chain
H: neutralizing antibody 22A12, heavy chain


Theoretical massNumber of molelcules
Total (without water)46,6112
Polyers46,6112
Non-polymers00
Water0
1
L: neutralizing antibody 22A12, light chain
H: neutralizing antibody 22A12, heavy chain
x 60


Theoretical massNumber of molelcules
Total (without water)2,796,653120
Polyers2,796,653120
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation60
2


  • idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
3
L: neutralizing antibody 22A12, light chain
H: neutralizing antibody 22A12, heavy chain
x 5


  • icosahedral pentamer
  • 233 kDa, 10 polymers
Theoretical massNumber of molelcules
Total (without water)233,05410
Polyers233,05410
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation5
4
L: neutralizing antibody 22A12, light chain
H: neutralizing antibody 22A12, heavy chain
x 6


  • icosahedral 23 hexamer
  • 280 kDa, 12 polymers
Theoretical massNumber of molelcules
Total (without water)279,66512
Polyers279,66512
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation6
5


  • idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1

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Components

#1: Protein/peptide neutralizing antibody 22A12, light chain / Fab22A12


Mass: 23261.770 Da / Num. of mol.: 1 / Fragment: Fab / Source: (natural) Mus musculus (house mouse)
#2: Protein/peptide neutralizing antibody 22A12, heavy chain / Fab22A12


Mass: 23349.105 Da / Num. of mol.: 1 / Fragment: Fab / Source: (natural) Mus musculus (house mouse)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeParent ID
1Fab fragment of 22A12 monoclonal antibody bound to Enterovirus 71 procapsid at the VP1 GH LoopCOMPLEX0
2Human enterovirus 71VIRUS1
3neutralizing antibody 22A12 Fab1
Details of virusEmpty: YES / Enveloped: NO / Virus host category: VERTEBRATES / Virus isolate: STRAIN / Virus type: VIRION
Natural hostOrganism: Homo sapiens / Strain: HeLa
Buffer solutionName: 10 mM Tris, 200 mM NaCl, 50 mM MgCl2 / Details: 10 mM Tris, 200 mM NaCl, 50 mM MgCl2 / pH: 7.5
SpecimenConc.: 1 / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: holey carbon Quantifoil EM grids
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE
Details: Plunged into ethane-propane mixture (FEI VITROBOT MARK III)

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TECNAI F20 / Date: Jul 15, 2011
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 50000 / Cs: 2 / Camera length: 0
Specimen holderSpecimen holder model: GATAN LIQUID NITROGEN
Image recordingFilm or detector model: KODAK SO-163 FILM
RadiationDiffraction protocol: SINGLE WAVELENGTH / Monochromatic or laue m l: M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameVersionCategory
1Situsmodel fitting
2UCSF Chimeramodel fitting
3Auto3DEM3D reconstruction
4EMAN13D reconstruction
5EMAN23D reconstruction
CTF correctionDetails: CTFFind3
SymmetryPoint symmetry: I
3D reconstructionMethod: Single Particle ReconstructionSingle particle analysis
Resolution: 8.8 / Resolution method: FSC 0.5 CUT-OFF / Number of particles: 15226 / Nominal pixel size: 1.27 / Actual pixel size: 1.27
Details: (Single particle details: Processing was completed with EMAN2 and AUTO3DEM) (Single particle--Applied symmetry: I)
Symmetry type: POINT
Atomic model buildingRef space: REAL
Atomic model buildingPDB-ID: 3GK8
Number of atoms included #LASTProtein: 3283 / Nucleic acid: 0 / Ligand: 0 / Solvent: 0 / Total: 3283

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