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- EMDB-6200: Cryo-electron microscopy of Enterovirus 71 (EV71) in complex with... -

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Entry
Database: EMDB / ID: 6200
TitleCryo-electron microscopy of Enterovirus 71 (EV71) in complex with Fab fragments of neutralizing antibody 22A12
Map dataReconstruction of EV71 procapsid in complex with Fab22A12
SampleFab fragment of 22A12 monoclonal antibody bound to Enterovirus 71 procapsid at the VP1 GH Loop:
virus / neutralizing antibody 22A12 Fab
KeywordsEV71 / picornavirus / Mab22A12 / antibody / Fab / neutralization / canyon / enterovirus / enterovirus 71
Function / homologyPeptidase C3, picornavirus core protein 2A / P-loop containing nucleoside triphosphate hydrolase / Helicase, superfamily 3, single-stranded RNA virus / Peptidase S1, PA clan / RNA-directed RNA polymerase, catalytic domain / AAA+ ATPase domain / Picornavirus coat protein VP4 / Picornavirus 2B protein / Picornavirus capsid / RNA-directed RNA polymerase, C-terminal domain ...Peptidase C3, picornavirus core protein 2A / P-loop containing nucleoside triphosphate hydrolase / Helicase, superfamily 3, single-stranded RNA virus / Peptidase S1, PA clan / RNA-directed RNA polymerase, catalytic domain / AAA+ ATPase domain / Picornavirus coat protein VP4 / Picornavirus 2B protein / Picornavirus capsid / RNA-directed RNA polymerase, C-terminal domain / Helicase, superfamily 3, single-stranded DNA/RNA virus / Peptidase C3A/C3B, picornaviral / Viral coat protein subunit / Picornavirus/Calicivirus coat protein / Poliovirus core protein 3a, soluble domain / picornavirus capsid protein / RNA dependent RNA polymerase / Superfamily 3 helicase of positive ssRNA viruses domain profile. / RdRp of positive ssRNA viruses catalytic domain profile. / Poliovirus 3A protein like / Picornavirus coat protein (VP4) / Picornavirus 2B protein / Picornavirus core protein 2A / RNA helicase / Poliovirus 3A protein-like / 3C cysteine protease (picornain 3C) / picornain 2A / pore-mediated entry of viral genome into host cell / suppression by virus of host mRNA export from nucleus / suppression by virus of host RIG-I activity / T=pseudo3 icosahedral viral capsid / picornain 3C / endocytosis involved in viral entry into host cell / RNA-protein covalent cross-linking / host cell cytoplasmic vesicle membrane / pore formation by virus in membrane of host cell / integral to membrane of host cell / RNA helicase activity / nucleoside-triphosphate phosphatase / RNA-directed RNA polymerase / suppression by virus of host gene expression / induction by virus of host autophagy / ion channel activity / protein complex oligomerization / viral RNA genome replication / RNA-directed 5'-3' RNA polymerase activity / cysteine-type endopeptidase activity / transcription, DNA-templated / virion attachment to host cell / structural molecule activity / RNA binding / membrane / ATP binding / Genome polyprotein
Function and homology information
SourceHuman enterovirus 71 (EV71) / unidentified (others)
Methodsingle particle reconstruction / cryo EM / 8.8 Å resolution
AuthorsShingler KL / Cifuente JO / Ashley RE / Makhov AM / Conway JF / 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 ReportPDB-ID: 3j91

SummaryFull report
PDB-ID: 3j93

SummaryFull report
About validation report
DateDeposition: Nov 21, 2014 / Header (metadata) release: Dec 10, 2014 / Map release: Dec 10, 2014 / Last update: Jan 28, 2015

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Structure visualization

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  • Surface view with section colored by density value
  • Surface level: 1
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  • Surface view colored by radius
  • Surface level: 1
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  • Surface view with fitted model
  • Atomic models: : PDB-3j91, PDB-3j93
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-3j91
  • Imaged by Jmol
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  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-3j93
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_6200.map.gz (map file in CCP4 format, 382705 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
461 pix
1.27 Å/pix.
= 585.47 Å
461 pix
1.27 Å/pix.
= 585.47 Å
461 pix
1.27 Å/pix.
= 585.47 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.27 Å
Density
Contour Level:1.0 (by author), 1 (movie #1):
Minimum - Maximum-4.29300165 - 8.48272610
Average (Standard dev.)0E-8 (1.00000000)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions461461461
Origin-230-230-230
Limit230230230
Spacing461461461
CellA=B=C: 585.47 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.271.271.27
M x/y/z461461461
origin x/y/z0.0000.0000.000
length x/y/z585.470585.470585.470
α/β/γ90.00090.00090.000
start NX/NY/NZ-72-72-72
NX/NY/NZ145145145
MAP C/R/S123
start NC/NR/NS-230-230-230
NC/NR/NS461461461
D min/max/mean-4.2938.483-0.000

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Supplemental data

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Sample components

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Entire Fab fragment of 22A12 monoclonal antibody bound to Enterovirus 71...

EntireName: Fab fragment of 22A12 monoclonal antibody bound to Enterovirus 71 procapsid at the VP1 GH Loop
Number of components: 2

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Component #1: virus, Human enterovirus 71

VirusName: Human enterovirus 71 / a.k.a: EV71Enterovirus 71 / Class: VIRION
Details: Virus procapsid was complexed with Fab fragments of neutralizing antibody 22A12.
Enveloped: No / Empty: Yes / Isolate: STRAIN
SpeciesSpecies: Human enterovirus 71 (EV71) / Strain: 1095/Shiga
Source (natural)Host Species: Homo sapiens (human) / Host category: VERTEBRATES

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Component #2: protein, neutralizing antibody 22A12 Fab

ProteinName: neutralizing antibody 22A12 Fab / Recombinant expression: No
SourceSpecies: unidentified (others)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 1 mg/ml / Buffer solution: 10 mM Tris, 200 mM NaCl, 50 mM MgCl2 / pH: 7.5
Support filmholey carbon Quantifoil EM grids
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE-PROPANE MIXTURE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20 / Date: Jul 15, 2011
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
LensMagnification: 50000 X (nominal) / Cs: 2 mm / Imaging mode: BRIGHT FIELD
Specimen HolderModel: GATAN LIQUID NITROGEN
CameraDetector: KODAK SO-163 FILM

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Image acquisition

Image acquisitionScanner: NIKON SUPER COOLSCAN 9000 / Sampling size: 6.35 microns

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: I (icosahedral) / Number of projections: 15226 / Details: Processing was completed with EMAN2 and AUTO3DEM.
3D reconstructionSoftware: EMAN, EMAN2, AUTO3DEM, Chimera / CTF correction: CTFFind3 / Resolution: 8.8 Å / Resolution method: FSC 0.5, semi-independent

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Atomic model buiding

Modeling #1Software: Situs / Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 4GMP
Output model

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