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- EMDB-0069: High-resolution Cryo-EM of Fab-labeled human parechovirus 3 -

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Basic information

Entry
Database: EMDB / ID: EMD-0069
TitleHigh-resolution Cryo-EM of Fab-labeled human parechovirus 3
Map dataHuman parechovirus type 3 in complex with neutralizing human monoclonal antibody Fabs Post-processed map, B factor -70
Sample
  • Complex: Human parechovirus type 3 in complex with fabs from AT12-015
    • Complex: fabs from AT12-015Semiconductor fabrication plant
      • Protein or peptide: AT12-015 antibody variable heavy
      • Protein or peptide: AT12-015 antibody variable light
    • Virus: Human parechovirus 3
      • RNA: RNA (5'-R(*UP*GP*GP*UP*AP*UP*UP*U)-3')
      • Protein or peptide: VP0
      • Protein or peptide: VP1
      • Protein or peptide: VP3
Function / homology
Function and homology information


RNA-protein covalent cross-linking / : / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / cytoplasmic vesicle membrane / viral capsid / : / protein complex oligomerization / monoatomic ion channel activity / RNA helicase activity ...RNA-protein covalent cross-linking / : / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / cytoplasmic vesicle membrane / viral capsid / : / protein complex oligomerization / monoatomic ion channel activity / RNA helicase activity / symbiont entry into host cell / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / DNA-templated transcription / virion attachment to host cell / structural molecule activity / RNA binding / ATP binding
Similarity search - Function
Viral polyprotein, parechovirus P3B / Parechovirus Genome-linked protein / Viral polyprotein, parechovirus P3A / Picornaviridae P3A protein / Helicase/polymerase/peptidase polyprotein, Calicivirus-type / Picornavirales 3C/3C-like protease domain / Picornavirales 3C/3C-like protease domain profile. / Peptidase C3A/C3B, picornaviral / 3C cysteine protease (picornain 3C) / Picornavirus capsid ...Viral polyprotein, parechovirus P3B / Parechovirus Genome-linked protein / Viral polyprotein, parechovirus P3A / Picornaviridae P3A protein / Helicase/polymerase/peptidase polyprotein, Calicivirus-type / 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. / 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
Capsid protein VP0 / Capsid protein VP0
Similarity search - Component
Biological speciesHomo sapiens (human) / Human parechovirus 3
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsDomanska A / Flatt JW / Jukonen JJJ / Geraets JA / Butcher SJ
Funding support Finland, 4 items
OrganizationGrant numberCountry
Academy of Finland275199 Finland
European UnionPIAPP-GA-2013-612308
European UnionPIEF-GA-2013-628150
European Union653706
CitationJournal: J Virol / Year: 2019
Title: A 2.8-Angstrom-Resolution Cryo-Electron Microscopy Structure of Human Parechovirus 3 in Complex with Fab from a Neutralizing Antibody.
Authors: Aušra Domanska / Justin W Flatt / Joonas J J Jukonen / James A Geraets / Sarah J Butcher /
Abstract: Human parechovirus 3 (HPeV3) infection is associated with sepsis characterized by significant immune activation and subsequent tissue damage in neonates. Strategies to limit infection have been ...Human parechovirus 3 (HPeV3) infection is associated with sepsis characterized by significant immune activation and subsequent tissue damage in neonates. Strategies to limit infection have been unsuccessful due to inadequate molecular diagnostic tools for early detection and the lack of a vaccine or specific antiviral therapy. Toward the latter, we present a 2.8-Å-resolution structure of HPeV3 in complex with fragments from a neutralizing human monoclonal antibody, AT12-015, using cryo-electron microscopy (cryo-EM) and image reconstruction. Modeling revealed that the epitope extends across neighboring asymmetric units with contributions from capsid proteins VP0, VP1, and VP3. Antibody decoration was found to block binding of HPeV3 to cultured cells. Additionally, at high resolution, it was possible to model a stretch of RNA inside the virion and, from this, identify the key features that drive and stabilize protein-RNA association during assembly. Human parechovirus 3 (HPeV3) is receiving increasing attention as a prevalent cause of sepsis-like symptoms in neonates, for which, despite the severity of disease, there are no effective treatments available. Structural and molecular insights into virus neutralization are urgently needed, especially as clinical cases are on the rise. Toward this goal, we present the first structure of HPeV3 in complex with fragments from a neutralizing monoclonal antibody. At high resolution, it was possible to precisely define the epitope that, when targeted, prevents virions from binding to cells. Such an atomic-level description is useful for understanding host-pathogen interactions and viral pathogenesis mechanisms and for finding potential cures for infection and disease.
History
DepositionJun 20, 2018-
Header (metadata) releaseJul 18, 2018-
Map releaseNov 21, 2018-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6gv4
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0069.png

Downloads & links

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Map

FileDownload / File: emd_0069.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHuman parechovirus type 3 in complex with neutralizing human monoclonal antibody Fabs Post-processed map, B factor -70
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.19998817 - 0.41895282
Average (Standard dev.)0.0015927622 (±0.024973061)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-240-240-240
Dimensions480480480
Spacing480480480
CellA=B=C: 508.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z480480480
origin x/y/z0.0000.0000.000
length x/y/z508.800508.800508.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-240-240-240
NC/NR/NS480480480
D min/max/mean-0.2000.4190.002

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Supplemental data

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Sample components

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Entire : Human parechovirus type 3 in complex with fabs from AT12-015

EntireName: Human parechovirus type 3 in complex with fabs from AT12-015
Components
  • Complex: Human parechovirus type 3 in complex with fabs from AT12-015
    • Complex: fabs from AT12-015Semiconductor fabrication plant
      • Protein or peptide: AT12-015 antibody variable heavy
      • Protein or peptide: AT12-015 antibody variable light
    • Virus: Human parechovirus 3
      • RNA: RNA (5'-R(*UP*GP*GP*UP*AP*UP*UP*U)-3')
      • Protein or peptide: VP0
      • Protein or peptide: VP1
      • Protein or peptide: VP3

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Supramolecule #1: Human parechovirus type 3 in complex with fabs from AT12-015

SupramoleculeName: Human parechovirus type 3 in complex with fabs from AT12-015
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 7.7 MDa

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Supramolecule #3: fabs from AT12-015

SupramoleculeName: fabs from AT12-015 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #5-#6
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Supramolecule #2: Human parechovirus 3

SupramoleculeName: Human parechovirus 3 / type: virus / ID: 2 / Parent: 1 / Macromolecule list: #1-#4 / NCBI-ID: 195055 / Sci species name: Human parechovirus 3 / Sci species strain: A308/99 / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No

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Macromolecule #1: RNA (5'-R(*UP*GP*GP*UP*AP*UP*UP*U)-3')

MacromoleculeName: RNA (5'-R(*UP*GP*GP*UP*AP*UP*UP*U)-3') / type: rna / ID: 1 / Details: RNA / Number of copies: 1
Source (natural)Organism: Human parechovirus 3 / Organ: colon adenocarcinoma
Molecular weightTheoretical: 2.505489 KDa
SequenceString:
UGGUAUUU

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Macromolecule #2: VP0

MacromoleculeName: VP0 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human parechovirus 3 / Organ: colon adenocarcinoma
Molecular weightTheoretical: 31.770135 KDa
SequenceString: MESIKDLVNV ATGAMDTLSL SNVETEVNNI ISGNEVGGEI ITKVADDASN LLGPNSFATT AQPENKDVVQ ATTTVNTTNL TQHPSAPTI PFTPDFKNVD NFHSMAYDIT TGDKNPSKLI RLDTASWQTS YSRQYEITTV ELPKSFWDDT RKPAYGQAKY F AAVRCGFH ...String:
MESIKDLVNV ATGAMDTLSL SNVETEVNNI ISGNEVGGEI ITKVADDASN LLGPNSFATT AQPENKDVVQ ATTTVNTTNL TQHPSAPTI PFTPDFKNVD NFHSMAYDIT TGDKNPSKLI RLDTASWQTS YSRQYEITTV ELPKSFWDDT RKPAYGQAKY F AAVRCGFH FQVQVNVNQG TAGSALVVYE PKPVIDSRQY LEFGSLTNLP HVLMNLAETT QADLCIPYVA DTNYVKTDSS DL GQLRVYV WTPLSVPTGA SNEVDVTVMG SLLQLDFQNP RPYGEDVEIY DN

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Macromolecule #3: VP1

MacromoleculeName: VP1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human parechovirus 3 / Organ: colon adenocarcinoma
Molecular weightTheoretical: 25.913127 KDa
SequenceString: NSWGSQMDLT DPLCIEDNME NCKQSISPNE LGLTSAQDDG PLGNEKPNYF LNFRTMNVDI FTVSHTKVDN IFGRAWYVTS HDFNNGDTW RQKLTFPKEG HGMLSQFFAY FTGEINIHIL YMAKQGFLRV AHTYDTEDNR KTFLSSNGVI TIPAGEQMTL S VPFYSNKP ...String:
NSWGSQMDLT DPLCIEDNME NCKQSISPNE LGLTSAQDDG PLGNEKPNYF LNFRTMNVDI FTVSHTKVDN IFGRAWYVTS HDFNNGDTW RQKLTFPKEG HGMLSQFFAY FTGEINIHIL YMAKQGFLRV AHTYDTEDNR KTFLSSNGVI TIPAGEQMTL S VPFYSNKP LRTVRHDSAL GFLMCRPSMH GTTRTTVEVY VSLRCPNFFF PVPAPKPTGS RATALSDESP Y

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Macromolecule #4: VP3

MacromoleculeName: VP3 / type: protein_or_peptide / ID: 4 / Details: polypeptide chain / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human parechovirus 3 / Organ: colon adenocarcinoma
Molecular weightTheoretical: 28.757551 KDa
SequenceString: GPNKANVSKF NKRKFLTAST KYKWTRTKVD IAEGPGTMNM ANVLSTTGAQ SVALVGERAF YDPRTAGSKS RFDDMIKIAQ LFSVMSDNT TPSSSSGIDK YGYFDWAATV APQNMVHRNV VTLDQFPNLN LFMNTYSYFR GSLIIRLSIY ASTFNRGRLR M GFFPNCTH ...String:
GPNKANVSKF NKRKFLTAST KYKWTRTKVD IAEGPGTMNM ANVLSTTGAQ SVALVGERAF YDPRTAGSKS RFDDMIKIAQ LFSVMSDNT TPSSSSGIDK YGYFDWAATV APQNMVHRNV VTLDQFPNLN LFMNTYSYFR GSLIIRLSIY ASTFNRGRLR M GFFPNCTH DTQLELDNAI YTICDIGSDN SFELTIPYSF STWMRKTHGH QLGLFQVEVL NRLTYNSSSP NKVHCIVQGR LG DDAKFFC PTGSLVSFQ

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Macromolecule #5: AT12-015 antibody variable heavy

MacromoleculeName: AT12-015 antibody variable heavy / type: protein_or_peptide / ID: 5 / Details: polypeptide chain / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.128613 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYAMSWVRQA PGKGLEWVSA ISGGGDSRYY ADSVKGRFTI SRDNSKNTLY LQMNSLGAE DTALYYCAKR LGRVAEYYFD YWGQGTLVTV SP

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Macromolecule #6: AT12-015 antibody variable light

MacromoleculeName: AT12-015 antibody variable light / type: protein_or_peptide / ID: 6 / Details: polypeptide chain / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 12.339752 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
DIQMTQSPST LSASVGDRVT ITCRTSQSIS NWLAWYQQKP GKAPKLLIYQ ASTLENGVPS RFTGSGSGTE FSLTISSLQP DDFATYYCQ QYNNYMALTF GGGTKVEIKR TVAA

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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

Concentration0.1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
10.0 mMTris-HClTristris hydrochloride
150.0 mMNaClSodium chloridesodium chloride
1.0 mMMgCLmagnesium chloride
GridMaterial: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE
Details: ultrathin carbon-coated lacey 400-mesh copper grids (Ted Pella product #01824)
VitrificationCryogen name: ETHANE / Chamber temperature: 295 K / Instrument: HOMEMADE PLUNGER
Details: We could not control humidity during plunging. It was ambient humidity. Blot for 1 s 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: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Digitization - Frames/image: 2-17 / Number grids imaged: 2 / Number real images: 6541 / Average exposure time: 1.0 sec. / Average electron dose: 48.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 217212
Details: automatic particle selection in RELION using template generated from manually selected particles
CTF correctionDetails: GCTF was used to estimate ctf
Startup modelType of model: OTHER
Details: Low resolution cryo-EM map done in AUTO3DEM from images of the same sample taken with FEI Tecnai TF20 on CCD camera
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.0)
Final 3D classificationNumber classes: 10 / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final reconstructionNumber classes used: 3 / Applied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 74927

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Atomic model buiding 1

DetailsInitial model was generated in I-TASSER and SWISSMODEL using 4z92 and 4udf as reference. Initial rigid fit of the model to the map was done in UCSF Chimera. Model refinement was done in Coot and MDFF.
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-6gv4:
High-resolution Cryo-EM of Fab-labeled human parechovirus 3

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