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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 dataThe cryoEM map of EV71 mature viron in complex with the Fab fragment of antibody D5
Sample
  • Sample: EV71 mature viron in complex with the Fab fragment of antibody D5
  • Virus: Human enterovirus 71
  • Protein or peptide: antibody D5
KeywordsEnterovirus 71(EV71) / virus-antibody complex / bivalent binding / high resolution cryo-EM
Function / homology
Function and homology information


RNA-protein covalent cross-linking / : / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MDA-5 activity / picornain 2A / symbiont-mediated suppression of host mRNA export from nucleus / symbiont genome entry into host cell via pore formation in plasma membrane / picornain 3C / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / endocytosis involved in viral entry into host cell ...RNA-protein covalent cross-linking / : / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MDA-5 activity / picornain 2A / symbiont-mediated suppression of host mRNA export from nucleus / symbiont genome entry into host cell via pore formation in plasma membrane / picornain 3C / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / endocytosis involved in viral entry into host cell / cytoplasmic vesicle membrane / : / viral capsid / nucleoside-triphosphate phosphatase / protein complex oligomerization / monoatomic ion channel activity / RNA helicase activity / induction by virus of host autophagy / RNA-directed RNA polymerase / symbiont entry into host cell / symbiont-mediated suppression of host gene expression / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / DNA-templated transcription / host cell nucleus / structural molecule activity / virion attachment to host cell / RNA binding / ATP binding / metal ion binding / nucleus
Similarity search - Function
Poliovirus 3A protein-like / Poliovirus 3A protein like / Picornavirus 2B protein / Poliovirus core protein 3a, soluble domain / Picornavirus 2B protein / Peptidase C3, picornavirus core protein 2A / Picornavirus core protein 2A / Picornavirus coat protein VP4 / Picornavirus coat protein (VP4) / Peptidase C3A/C3B, picornaviral ...Poliovirus 3A protein-like / Poliovirus 3A protein like / Picornavirus 2B protein / Poliovirus core protein 3a, soluble domain / Picornavirus 2B protein / Peptidase C3, picornavirus core protein 2A / Picornavirus core protein 2A / Picornavirus coat protein VP4 / Picornavirus coat protein (VP4) / Peptidase C3A/C3B, picornaviral / 3C cysteine protease (picornain 3C) / Picornavirus capsid / picornavirus capsid protein / Helicase, superfamily 3, single-stranded RNA virus / Superfamily 3 helicase of positive ssRNA viruses domain profile. / Helicase, superfamily 3, single-stranded DNA/RNA virus / RNA helicase / Picornavirus/Calicivirus coat protein / Viral coat protein subunit / RNA-directed RNA polymerase, C-terminal domain / Viral RNA-dependent RNA polymerase / Reverse transcriptase/Diguanylate cyclase domain / RNA-directed RNA polymerase, catalytic domain / RdRp of positive ssRNA viruses catalytic domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Genome polyprotein / Genome polyprotein
Similarity search - Component
Biological speciesHomo sapiens (human) / Human enterovirus 71
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.
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)
AnnotationThe cryoEM map of EV71 mature viron in complex with the Fab fragment of antibody D5
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
Components
  • Sample: EV71 mature viron in complex with the Fab fragment of antibody D5
  • Virus: Human enterovirus 71
  • Protein or peptide: antibody D5

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Supramolecule #1000: EV71 mature viron in complex with the Fab fragment of antibody D5

SupramoleculeName: EV71 mature viron in complex with the Fab fragment of antibody D5
type: sample / ID: 1000
Oligomeric state: One fab fragment of antibody D5 bind to one protomer of EV71
Number unique components: 2
Molecular weightTheoretical: 8 MDa

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Supramolecule #1: Human enterovirus 71

SupramoleculeName: Human enterovirus 71 / type: virus / ID: 1
Details: EV71 mature viron in complex with the Fab fragment of antibody D5
NCBI-ID: 39054 / Sci species name: Human enterovirus 71 / Sci species strain: G082 / Database: NCBI / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Homo sapiens (human) / synonym: VERTEBRATES
Molecular weightTheoretical: 5 MDa
Virus shellShell ID: 1 / Diameter: 300 Å / T number (triangulation number): 3

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Macromolecule #1: antibody D5

MacromoleculeName: antibody D5 / type: protein_or_peptide / ID: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Homo sapiens (human) / synonym: Human

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.8 mg/mL
BufferpH: 7.6 / Details: 0.15 M phosphate buffered saline(PBS) buffer
StainingType: NEGATIVE
Details: Grids were plunge-frozen into liquid ethane using a FEI Mark IV virtobot, after 2s blotting to remove extra sample.
GridDetails: 200 mesh R1.2x1.3 Quantifoil Cu grid, glow discharged in air.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK IV / Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.005 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 37000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureMin: 90 K / Max: 92 K / Average: 91 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 75000 magnification
DateOct 2, 2014
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 320 / Average electron dose: 16 e/Å2
Details: Every image is the average of seven frames recorded by the direct electron detector
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 4.8 Å / Resolution method: OTHER / Software - Name: jspr, EMAN, EMAN2 / Number images used: 2902
DetailsThe 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.

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Atomic model buiding 1

Initial modelPDB ID:

Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3jau:
The cryoEM map of EV71 mature viron in complex with the Fab fragment of antibody D5

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