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- EMDB-6964: Fit R10 Fab coordinates into the cryo-EM of EV71 in complex with A9 -

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Basic information

Entry
Database: EMDB / ID: EMD-6964
TitleFit R10 Fab coordinates into the cryo-EM of EV71 in complex with A9
Map data
Sample
  • Complex: Complex of EV71 and A9 neutralizing antibody Fab
    • Complex: EV71Enterovirus 71
      • Protein or peptide: Capsid protein VP1
      • Protein or peptide: VP2
      • Protein or peptide: VP3
    • Complex: A9 neutralizing antibody Fab
      • Protein or peptide: R10 ANTIBODY LIGHT CHAIN
      • Protein or peptide: R10 ANTIBODY HEAVY CHAIN
Function / homology
Function and homology information


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 / cytoplasmic vesicle membrane / endocytosis involved in viral entry into host cell / viral capsid ...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 / cytoplasmic vesicle membrane / endocytosis involved in viral entry into host cell / viral capsid / : / nucleoside-triphosphate phosphatase / protein complex oligomerization / monoatomic ion channel activity / host cell cytoplasm / RNA helicase activity / induction by virus of host autophagy / symbiont entry into host cell / RNA-directed RNA polymerase / symbiont-mediated suppression of host gene expression / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / DNA-templated transcription / virion attachment to host cell / structural molecule activity / ATP hydrolysis activity / proteolysis / RNA binding / ATP binding / metal ion binding / cytoplasm
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) / Picornavirales 3C/3C-like protease domain ...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) / Picornavirales 3C/3C-like protease domain / Picornavirales 3C/3C-like protease domain profile. / 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 / Genome polyprotein / Genome polyprotein
Similarity search - Component
Biological speciesEnterovirus A71 / Mus musculoides (Temminck's mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.8 Å
AuthorsWang X / Zhu L / Wang N
CitationJournal: mBio / Year: 2018
Title: Neutralization Mechanisms of Two Highly Potent Antibodies against Human Enterovirus 71.
Authors: Ling Zhu / Kangwei Xu / Nan Wang / Lei Cao / Junlan Wu / Qiang Gao / Elizabeth E Fry / David I Stuart / Zihe Rao / Junzhi Wang / Xiangxi Wang /
Abstract: Despite significant advances in health care, outbreaks of infections by enteroviruses (EVs) continue to plague the Asia-Pacific region every year. Enterovirus 71 (EV71) causes hand-foot-and-mouth ...Despite significant advances in health care, outbreaks of infections by enteroviruses (EVs) continue to plague the Asia-Pacific region every year. Enterovirus 71 (EV71) causes hand-foot-and-mouth disease (HFMD), for which there are currently no therapeutics. Here, we report two new antibodies, A9 and D6, that potently neutralize EV71. A9 exhibited a 50% neutralizing concentration (neut) value of 0.1 nM against EV71, which was 10-fold lower than that observed for D6. Investigation into the mechanisms of neutralization revealed that binding of A9 to EV71 blocks receptor binding but also destabilizes and damages the virus capsid structure. In contrast, D6 destabilizes the capsid only slightly but interferes more potently with the attachment of the virus to the host cells. Cryo-electron microscopy (cryo-EM) structures of A9 and D6 bound with EV71 shed light on the locations and nature of the epitopes recognized by the two antibodies. Although some regions of the epitopes recognized by the two antibodies overlap, there are differences that give rise to dissimilarities in potency as well as in the mechanisms of neutralization. Interestingly, the overlapping regions of the epitopes encompass the site that the virus uses to bind SCARB2, explaining the reason for the observed blocking of the virus-receptor interaction by the two antibodies. We also identified structural elements that might play roles in modulating the stability of the EV71 particles, including particle integrity. The molecular features of the A9 and D6 epitopes unveiled in this study open up new avenues for rationally designing antiviral drugs. During the course of viral infections, the human body produces neutralizing antibodies which play a defining role in clearing the virus. From this study, we report two new, highly potent neutralizing antibodies, A9 and D6, against enterovirus 71 (EV71), the causative agent of HFMD. Both antibodies prevent the virus from entering the host cell, a step that is important for establishing a successful infection. A9 destabilizes and damages the virus capsid that forms an outer protective covering around the genome of the virus, while also interfering with virus attachment to the host cells. In contrast, D6 only prevents binding of the virus to its receptor(s). The mechanism of neutralization of A9 is unique and has not been observed before for neutralizing antibodies targeting EVs. The two antibodies that we are reporting in this study have potential to be developed into much-needed therapeutic interventions for treatment of HFMD, outbreaks of which are reported every year in the Asia-Pacific region.
History
DepositionMay 7, 2018-
Header (metadata) releaseDec 25, 2019-
Map releaseDec 25, 2019-
UpdateJan 6, 2021-
Current statusJan 6, 2021Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5zuf
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5zuf
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6964.png

Downloads & links

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Map

FileDownload / File: emd_6964.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.12881522 - 0.18014663
Average (Standard dev.)0.00038155378 (±0.010382689)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 648.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z480480480
origin x/y/z0.0000.0000.000
length x/y/z648.000648.000648.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS480480480
D min/max/mean-0.1290.1800.000

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Supplemental data

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Sample components

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Entire : Complex of EV71 and A9 neutralizing antibody Fab

EntireName: Complex of EV71 and A9 neutralizing antibody Fab
Components
  • Complex: Complex of EV71 and A9 neutralizing antibody Fab
    • Complex: EV71Enterovirus 71
      • Protein or peptide: Capsid protein VP1
      • Protein or peptide: VP2
      • Protein or peptide: VP3
    • Complex: A9 neutralizing antibody Fab
      • Protein or peptide: R10 ANTIBODY LIGHT CHAIN
      • Protein or peptide: R10 ANTIBODY HEAVY CHAIN

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Supramolecule #1: Complex of EV71 and A9 neutralizing antibody Fab

SupramoleculeName: Complex of EV71 and A9 neutralizing antibody Fab / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: EV71

SupramoleculeName: EV71 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#3
Source (natural)Organism: Enterovirus A71
Recombinant expressionOrganism: Chlorocebus aethiops (grivet)

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Supramolecule #3: A9 neutralizing antibody Fab

SupramoleculeName: A9 neutralizing antibody Fab / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #4-#5
Source (natural)Organism: Mus musculoides (Temminck's mouse)
Recombinant expressionOrganism: Mus musculoides (Temminck's mouse)

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Macromolecule #1: Capsid protein VP1

MacromoleculeName: Capsid protein VP1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Enterovirus A71
Molecular weightTheoretical: 32.787902 KDa
Recombinant expressionOrganism: Chlorocebus aethiops (grivet)
SequenceString: GDRVADVIES SIEDSSSRAL THALPAPTGQ NTQVSSHRLD TGKVPALQAA EIGASSNASD ESMIETRCVL NSHSTAETTL DSFFSRAGL VGEIDLPLKG TTNPNGYANW DIDITGYAQM RRKVELFTYM RFDAEFTFVA CTPTGEVVPQ LLQYMFVPPG A PKPDSRES ...String:
GDRVADVIES SIEDSSSRAL THALPAPTGQ NTQVSSHRLD TGKVPALQAA EIGASSNASD ESMIETRCVL NSHSTAETTL DSFFSRAGL VGEIDLPLKG TTNPNGYANW DIDITGYAQM RRKVELFTYM RFDAEFTFVA CTPTGEVVPQ LLQYMFVPPG A PKPDSRES LAWQTATNPS VFVKLSDPPA QVSVPFMSPA SAYQWFYDGY PTFGEHKQEK DLEYGAMPNN MMGTFSVRTV GT SKSKYPL VVRIYMRMKH VRAWIPRPMR NQNYLFKANP NYAGNSIKPT GASRTAITTL

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Macromolecule #2: VP2

MacromoleculeName: VP2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Enterovirus A71
Molecular weightTheoretical: 26.874252 KDa
Recombinant expressionOrganism: Chlorocebus aethiops (grivet)
SequenceString: SDRVAQLTIG NSTITTQEAA NIIVGYGEWP SYCSDSDATA VDKPTRPDVS VNRFYTLDTK LWEKSSKGWY WKFPDVLTET GVFGQNAQF HYLYRSGFCI HVQCNASKFH QGALLVAVLP EYVIGTVAGG TGTEDTHPPY KQTQPGADGF ELQHPYVLDA G IPISQLTV ...String:
SDRVAQLTIG NSTITTQEAA NIIVGYGEWP SYCSDSDATA VDKPTRPDVS VNRFYTLDTK LWEKSSKGWY WKFPDVLTET GVFGQNAQF HYLYRSGFCI HVQCNASKFH QGALLVAVLP EYVIGTVAGG TGTEDTHPPY KQTQPGADGF ELQHPYVLDA G IPISQLTV CPHQWINLRT NNCATIIVPY INALPFDSAL NHCNFGLLVV PISPLDYDQG ATPVIPITIT LAPMCSEFAG LR QAVTQ

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Macromolecule #3: VP3

MacromoleculeName: VP3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Enterovirus A71
Molecular weightTheoretical: 26.468225 KDa
Recombinant expressionOrganism: Chlorocebus aethiops (grivet)
SequenceString: GFPTELKPGT NQFLTTDDGV SAPILPNFHP TPCIHIPGEV RNLLELCQVE TILEVNNVPT NATSLMERLR FPVSAQAGKG ELCAVFRAD PGRNGPWQST LLGQLCGYYT QWSGSLEVTF MFTGSFMATG KMLIAYTPPG GPLPKDRATA MLGTHVIWDF G LQSSVTLV ...String:
GFPTELKPGT NQFLTTDDGV SAPILPNFHP TPCIHIPGEV RNLLELCQVE TILEVNNVPT NATSLMERLR FPVSAQAGKG ELCAVFRAD PGRNGPWQST LLGQLCGYYT QWSGSLEVTF MFTGSFMATG KMLIAYTPPG GPLPKDRATA MLGTHVIWDF G LQSSVTLV IPWISNTHYR AHARDGVFDY YTTGLVSIWY QTNYVVPIGA PNTAYIIALA AAQKNFTMKL CKDASDILQT GT IQ

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Macromolecule #4: R10 ANTIBODY LIGHT CHAIN

MacromoleculeName: R10 ANTIBODY LIGHT CHAIN / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculoides (Temminck's mouse)
Molecular weightTheoretical: 23.283535 KDa
Recombinant expressionOrganism: Mus musculoides (Temminck's mouse)
SequenceString: DIVLTQSPAI MSASPGEKVT MTCSASSSVS YIHWYQQKSG TSPKRWIYDT SRLAFGVPGR FSGSGSGTSY SLTISSMEAE DAATYYCQQ WSSNYTFGGG TNLEIKRADA APTVSIFPPS SEQLTSGGAS VVCFLNNFYP KDINVKWKID GSERQNGVLN S WTDQDSKD ...String:
DIVLTQSPAI MSASPGEKVT MTCSASSSVS YIHWYQQKSG TSPKRWIYDT SRLAFGVPGR FSGSGSGTSY SLTISSMEAE DAATYYCQQ WSSNYTFGGG TNLEIKRADA APTVSIFPPS SEQLTSGGAS VVCFLNNFYP KDINVKWKID GSERQNGVLN S WTDQDSKD STYSMSSTLT LTKDEYERHN SYTCEATHKT STSPIVKSFN RNEC

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Macromolecule #5: R10 ANTIBODY HEAVY CHAIN

MacromoleculeName: R10 ANTIBODY HEAVY CHAIN / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculoides (Temminck's mouse)
Molecular weightTheoretical: 23.67165 KDa
Recombinant expressionOrganism: Mus musculoides (Temminck's mouse)
SequenceString: EVKLVESGGG SVKPGGSLKL SCAASGFSFS TYGMSWVRQT PEKRLEWVAT ISGGGGYTYY PDSVKGRFTI SRDNARNILY LQMSSLRSG DTAMYYCARR VTTVAEYYFD YWGQGTTLTV SSPKTTPPSV YPLAPAAAQT NSMVTLGCLV KGYFPEPVTV T WNSGSLSS ...String:
EVKLVESGGG SVKPGGSLKL SCAASGFSFS TYGMSWVRQT PEKRLEWVAT ISGGGGYTYY PDSVKGRFTI SRDNARNILY LQMSSLRSG DTAMYYCARR VTTVAEYYFD YWGQGTTLTV SSPKTTPPSV YPLAPAAAQT NSMVTLGCLV KGYFPEPVTV T WNSGSLSS GVHTFPAVLQ SDLYTLSSSV TVPSSTWPSE TVTCNVAHPA SSTKVDKKIV PR

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 20.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1100

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