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- EMDB-6366: The cryoEM map of EV71 mature viron in complex with the Fab fragm... -

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Basic information

Entry
Database: EMDB / ID: EMD-6366
TitleThe cryoEM map of EV71 mature viron in complex with the Fab fragment of antibody D5
Map data
SampleEV71 mature viron in complex with the Fab fragment of antibody D5:
virus / antibody D5
KeywordsEnterovirus 71(EV71) / virus-antibody complex / bivalent binding / high resolution cryo-EM
Function / homology
Function and homology information


suppression by virus of host MDA-5 activity / picornain 2A / pore-mediated entry of viral genome into host cell / suppression by virus of host mRNA export from nucleus / picornain 3C / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / RNA-protein covalent cross-linking / integral to membrane of host cell / pore formation by virus in membrane of host cell ...suppression by virus of host MDA-5 activity / picornain 2A / pore-mediated entry of viral genome into host cell / suppression by virus of host mRNA export from nucleus / picornain 3C / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / RNA-protein covalent cross-linking / integral to membrane of host cell / pore formation by virus in membrane of host cell / viral capsid / endocytosis involved in viral entry into host cell / protein complex oligomerization / cytoplasmic vesicle membrane / nucleoside-triphosphate phosphatase / ion channel activity / suppression by virus of host gene expression / induction by virus of host autophagy / RNA-directed RNA polymerase / viral entry into host cell / cysteine-type endopeptidase activity / viral RNA genome replication / RNA-directed 5'-3' RNA polymerase activity / RNA helicase activity / transcription, DNA-templated / virion attachment to host cell / host cell nucleus / structural molecule activity / RNA binding / ATP binding / metal ion binding / nucleus
Picornavirus capsid / Peptidase S1, PA clan / Picornavirus 2B protein / AAA+ ATPase domain / Picornavirus coat protein VP4 / Helicase, superfamily 3, single-stranded RNA virus / Peptidase C3, picornavirus core protein 2A / Peptidase C3A/C3B, picornaviral / Poliovirus 3A protein-like / P-loop containing nucleoside triphosphate hydrolase ...Picornavirus capsid / Peptidase S1, PA clan / Picornavirus 2B protein / AAA+ ATPase domain / Picornavirus coat protein VP4 / Helicase, superfamily 3, single-stranded RNA virus / Peptidase C3, picornavirus core protein 2A / Peptidase C3A/C3B, picornaviral / Poliovirus 3A protein-like / P-loop containing nucleoside triphosphate hydrolase / Viral coat protein subunit / Helicase, superfamily 3, single-stranded DNA/RNA virus / Picornavirus/Calicivirus coat protein / Poliovirus core protein 3a, soluble domain / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily / Peptidase S1, PA clan, chymotrypsin-like fold / RNA-directed RNA polymerase, C-terminal domain / RNA-directed RNA polymerase, catalytic domain
Genome polyprotein / Genome polyprotein
Biological speciesHuman enterovirus 71 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 4.8 Å
AuthorsFan C / Ye XH / Ku ZQ / Zuo T / Kong LL / Zhang C / Shi JP / Liu QW / Chen T / Zhang YY ...Fan C / Ye XH / Ku ZQ / Zuo T / Kong LL / Zhang C / Shi JP / Liu QW / Chen T / Zhang YY / Jiang W / Zhang LQ / Huang Z / Cong Y
CitationJournal: PLoS Pathog / Year: 2016
Title: Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody.
Authors: Xiaohua Ye / Chen Fan / Zhiqiang Ku / Teng Zuo / Liangliang Kong / Chao Zhang / Jinping Shi / Qingwei Liu / Tan Chen / Yingyi Zhang / Wen Jiang / Linqi Zhang / Zhong Huang / Yao Cong /
Abstract: Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly ...Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionJun 24, 2015-
Header (metadata) releaseNov 25, 2015-
Map releaseFeb 10, 2016-
UpdateMay 25, 2016-
Current statusMay 25, 2016Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jau
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3jau
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_6366.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.79 Å/pix.
x 320 pix.
= 572.8 Å
1.79 Å/pix.
x 320 pix.
= 572.8 Å
1.79 Å/pix.
x 320 pix.
= 572.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.79 Å
Density
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.5
Minimum - Maximum-1.67944169 - 1.87832439
Average (Standard dev.)-0.00001208 (±0.18172173)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-160-160-160
Dimensions320320320
Spacing320320320
CellA=B=C: 572.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.791.791.79
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z572.800572.800572.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-160-160-160
NC/NR/NS320320320
D min/max/mean-1.6791.878-0.000

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Supplemental data

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Sample components

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Entire EV71 mature viron in complex with the Fab fragment of antibody D5

EntireName: EV71 mature viron in complex with the Fab fragment of antibody D5
Number of components: 2
Oligomeric State: One fab fragment of antibody D5 bind to one protomer of EV71
MassTheoretical: 8 MDa

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Component #1: virus, Human enterovirus 71

VirusName: Human enterovirus 71 / Class: VIRION
Details: EV71 mature viron in complex with the Fab fragment of antibody D5
Enveloped: No / Empty: No / Isolate: STRAIN
MassTheoretical: 5 MDa
SpeciesSpecies: Human enterovirus 71 / Strain: G082
Source (natural)Host Species: Homo sapiens (human) / Host category: VERTEBRATES

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Component #2: protein, antibody D5

ProteinName: antibody D5 / Recombinant expression: No
SourceSpecies: Homo sapiens (human)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: negative staining, cryo EM
Sample solutionSpecimen conc.: 0.8 mg/mL
Buffer solution: 0.15 M phosphate buffered saline(PBS) buffer
pH: 7.6
Support film200 mesh R1.2x1.3 Quantifoil Cu grid, glow discharged in air.
StainingGrids were plunge-frozen into liquid ethane using a FEI Mark IV virtobot, after 2s blotting to remove extra sample.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 120 K / Humidity: 100 % / Method: Blot for 2 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Oct 2, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 16 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 37000 X (nominal)
Astigmatism: Objective lens astigmatism was corrected at 75000 magnification
Cs: 0.005 mm / Imaging mode: BRIGHT FIELD / Defocus: 2000 - 3500 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: 91 (90 - 92 K)
CameraDetector: FEI FALCON II (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 320
Details: Every image is the average of seven frames recorded by the direct electron detector

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: I (icosahedral) / Number of projections: 2902
Details: The particles were boxed using e2boxer.py. CTF fitting was automatically performed using fitctf2.py in jspr, then visually validated and adjusted using EMAN1.9 ctfit program. The gold ...Details: The particles were boxed using e2boxer.py. CTF fitting was automatically performed using fitctf2.py in jspr, then visually validated and adjusted using EMAN1.9 ctfit program. The gold standard 3D reconstruction procedure was followed using jspr package, with the datasets split into two halves in the beginning.
3D reconstructionAlgorithm: common lines / Software: jspr, EMAN, EMAN2 / CTF correction: Each particle / Resolution: 4.8 Å / Resolution method: FSC 0.143, gold-standard

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Atomic model buiding

Modeling #1Software: Chimera / Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 3VBS
Chain ID: A, B, C, D
Output model

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