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- PDB-3jau: The cryoEM map of EV71 mature viron in complex with the Fab fragm... -

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Entry
Database: PDB / ID: 3jau
TitleThe cryoEM map of EV71 mature viron in complex with the Fab fragment of antibody D5
Components
  • Capsid protein VP1
  • Heavy chain of Fab fragment variable region of antibody D5
  • Light chain of Fab fragment variable region of antibody D5
KeywordsVIRUS/IMMUNE SYSTEM / Enterovirus 71(EV71) / virus-antibody complex / bivalent binding / high resolution cryo-EM / VIRUS-IMMUNE SYSTEM complex
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 speciesHuman enterovirus
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsFan, C. / Ye, X.H. / Ku, Z.Q. / Zuo, T. / Kong, L.L. / Zhang, C. / Shi, J.P. / Liu, Q.W. / Chen, T. / Zhang, Y.Y. ...Fan, C. / Ye, X.H. / Ku, Z.Q. / Zuo, T. / Kong, L.L. / Zhang, C. / Shi, J.P. / Liu, Q.W. / Chen, T. / Zhang, Y.Y. / Jiang, W. / Zhang, L.Q. / 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, 2015Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Feb 10, 2016Provider: repository / Type: Initial release
Revision 1.1Oct 26, 2016Group: Database references / Source and taxonomy
Revision 1.2Dec 18, 2019Group: Data collection / Database references / Category: database_2 / em_image_scans / em_software

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Structure visualization

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  • Biological unit as complete icosahedral assembly
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  • Biological unit as icosahedral 23 hexamer
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  • Superimposition on EM map
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Assembly

Deposited unit
A: Capsid protein VP1
H: Heavy chain of Fab fragment variable region of antibody D5
L: Light chain of Fab fragment variable region of antibody D5


Theoretical massNumber of molelcules
Total (without water)27,1403
Polymers27,1403
Non-polymers00
Water0
1
A: Capsid protein VP1
H: Heavy chain of Fab fragment variable region of antibody D5
L: Light chain of Fab fragment variable region of antibody D5
x 60


  • complete icosahedral assembly
  • 180-MERIC
  • 1.63 MDa, 180 polymers
Theoretical massNumber of molelcules
Total (without water)1,628,395180
Polymers1,628,395180
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
A: Capsid protein VP1
H: Heavy chain of Fab fragment variable region of antibody D5
L: Light chain of Fab fragment variable region of antibody D5
x 5


  • icosahedral pentamer
  • 136 kDa, 15 polymers
Theoretical massNumber of molelcules
Total (without water)135,70015
Polymers135,70015
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation5
4
A: Capsid protein VP1
H: Heavy chain of Fab fragment variable region of antibody D5
L: Light chain of Fab fragment variable region of antibody D5
x 6


  • icosahedral 23 hexamer
  • 163 kDa, 18 polymers
Theoretical massNumber of molelcules
Total (without water)162,83918
Polymers162,83918
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
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Protein/peptide Capsid protein VP1 /


Mass: 2001.133 Da / Num. of mol.: 1 / Fragment: UNP residues 207-223 / Source method: isolated from a natural source / Source: (natural) Human enterovirus / References: UniProt: X2L816, UniProt: Q5DW45*PLUS
#2: Antibody Heavy chain of Fab fragment variable region of antibody D5


Mass: 13026.259 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mus musculus
#3: Antibody Light chain of Fab fragment variable region of antibody D5


Mass: 12112.521 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mus musculus

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: EV71 mature viron in complex with the Fab fragment of antibody D5
Type: COMPLEX
Details: One fab fragment of antibody D5 bind to one protomer of EV71
Molecular weightValue: 8 MDa / Experimental value: NO
Details of virusEmpty: NO / Enveloped: NO / Host category: VERTEBRATES / Isolate: STRAIN / Type: VIRION
Natural hostOrganism: Homo sapiens
Buffer solutionpH: 7.6 / Details: 0.15 M phosphate buffered saline(PBS) buffer
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 0.15 M phosphate buffered saline(PBS) buffer (Stain Details Grids were plunge-frozen into liquid ethane using a FEI Mark IV virtobot, after 2s blotting to remove extra sample.)
Specimen supportDetails: 200 mesh R1.2x1.3 Quantifoil Cu grid, glow discharged in air
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temp: 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
MicroscopyModel: FEI TITAN KRIOS / Date: Oct 2, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 37000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 2000 nm / Cs: 0.005 mm
Astigmatism: Objective lens astigmatism was corrected at 75000 magnification
Camera length: 0 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: 91 K / Temperature (max): 92 K / Temperature (min): 90 K / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 16 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k)

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Processing

EM software
IDNameCategory
1UCSF Chimeramodel fitting
2EMAN3D reconstruction
3jspr3D reconstruction
CTF correctionDetails: Each particle
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionMethod: common lines / Resolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2902
Details: (Single particle 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 ...Details: (Single particle 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.) (Single particle--Applied symmetry: I)
Refinement type: HALF-MAPS REFINED INDEPENDENTLY / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Details: REFINEMENT PROTOCOL--rigid body
Atomic model buildingPDB-ID: 3VBS

3vbs


Pdb chain-ID: D
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms1915 0 0 0 1915

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