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- EMDB-3138: Multiple capsid-stabilizing protein-RNA and protein-protein inter... -

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Basic information

Entry
Database: EMDB / ID: EMD-3138
TitleMultiple capsid-stabilizing protein-RNA and protein-protein interactions revealed in a high-resolution structure of an emerging picornavirus causing neonatal sepsis
Map dataReconstruction of human parechovirus 3 in complex with Fab AT12-015.
Sample
  • Sample: Fab fragment of Mab AT12-015 to Human Parechovirus 3
  • Virus: Human parechovirus 3
  • Protein or peptide: human monoclonal antibody AT12-015
Keywordspicornavirus / parechovirus / human parechovirus 3 / HPeV3 / neonatal sepsis / cryoEM / image processing / single particle anaylsis / Fab AT12-015 / HPeV3-Fab AT12-015
Biological speciesHomo sapiens (human) / Human parechovirus 3
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 15.0 Å
AuthorsShakeel S / Westerhuis BM / Domanska A / Koning RI / Matadeen R / Koster AJ / Bakker AQ / Beaumont T / Wolthers KC / Butcher SJ
CitationJournal: Nat Commun / Year: 2016
Title: Multiple capsid-stabilizing interactions revealed in a high-resolution structure of an emerging picornavirus causing neonatal sepsis.
Authors: Shabih Shakeel / Brenda M Westerhuis / Ausra Domanska / Roman I Koning / Rishi Matadeen / Abraham J Koster / Arjen Q Bakker / Tim Beaumont / Katja C Wolthers / Sarah J Butcher /
Abstract: The poorly studied picornavirus, human parechovirus 3 (HPeV3) causes neonatal sepsis with no therapies available. Our 4.3-Å resolution structure of HPeV3 on its own and at 15 Å resolution in ...The poorly studied picornavirus, human parechovirus 3 (HPeV3) causes neonatal sepsis with no therapies available. Our 4.3-Å resolution structure of HPeV3 on its own and at 15 Å resolution in complex with human monoclonal antibody Fabs demonstrates the expected picornavirus capsid structure with three distinct features. First, 25% of the HPeV3 RNA genome in 60 sites is highly ordered as confirmed by asymmetric reconstruction, and interacts with conserved regions of the capsid proteins VP1 and VP3. Second, the VP0 N terminus stabilizes the capsid inner surface, in contrast to other picornaviruses where on expulsion as VP4, it forms an RNA translocation channel. Last, VP1's hydrophobic pocket, the binding site for the antipicornaviral drug, pleconaril, is blocked and thus inappropriate for antiviral development. Together, these results suggest a direction for development of neutralizing antibodies, antiviral drugs based on targeting the RNA-protein interactions and dissection of virus assembly on the basis of RNA nucleation.
History
DepositionSep 3, 2015-
Header (metadata) releaseJan 13, 2016-
Map releaseAug 10, 2016-
UpdateAug 24, 2016-
Current statusAug 24, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5000
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 5000
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3138.tif

Downloads & links

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Map

FileDownload / File: emd_3138.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationReconstruction of human parechovirus 3 in complex with Fab AT12-015.
Voxel sizeX=Y=Z: 5.6 Å
Density
Contour LevelBy AUTHOR: 5000.0 / Movie #1: 5000
Minimum - Maximum-32768.0 - 32767.0
Average (Standard dev.)355.384674070000017 (±4700.456542969999646)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-50-50-50
Dimensions101101101
Spacing101101101
CellA=B=C: 565.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z5.65.65.6
M x/y/z101101101
origin x/y/z0.0000.0000.000
length x/y/z565.600565.600565.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-50-50-50
NC/NR/NS101101101
D min/max/mean-32768.00032767.000355.385

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Supplemental data

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Sample components

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Entire : Fab fragment of Mab AT12-015 to Human Parechovirus 3

EntireName: Fab fragment of Mab AT12-015 to Human Parechovirus 3
Components
  • Sample: Fab fragment of Mab AT12-015 to Human Parechovirus 3
  • Virus: Human parechovirus 3
  • Protein or peptide: human monoclonal antibody AT12-015

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Supramolecule #1000: Fab fragment of Mab AT12-015 to Human Parechovirus 3

SupramoleculeName: Fab fragment of Mab AT12-015 to Human Parechovirus 3 / type: sample / ID: 1000 / Details: The sample was monodisperse
Oligomeric state: icosahedrally-symmetric virus with 60 copies each of VP0, VP3 and VP1 in complex with 60 copies of Fab AT12-015
Number unique components: 2

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Supramolecule #1: Human parechovirus 3

SupramoleculeName: Human parechovirus 3 / type: virus / ID: 1 / Name.synonym: HPeV3 / NCBI-ID: 195055 / Sci species name: Human parechovirus 3 / Virus type: VIRION / Virus isolate: OTHER / Virus enveloped: No / Virus empty: No / Syn species name: HPeV3
Host (natural)Organism: Homo sapiens (human) / synonym: VERTEBRATES
Host systemOrganism: Chlorocebus aethiops (grivet) / Recombinant cell: Vero
Virus shellShell ID: 1 / Diameter: 280 Å / T number (triangulation number): 1

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Macromolecule #1: human monoclonal antibody AT12-015

MacromoleculeName: human monoclonal antibody AT12-015 / type: protein_or_peptide / ID: 1 / Name.synonym: mAb AT12-015
Details: Fab fragments of human monoclonal antibody AT12-015.
Number of copies: 60 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293

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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

Concentration1 mg/mL
BufferpH: 7.5 / Details: 10 mM Tris-HCL, 150 mM NaCl, 1 mM MgCl2
StainingType: NEGATIVE / Details: Unstained
GridDetails: Holey carbon copper grids with ultrathin carbon support from TED PELLA.
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 3.56 µm / Nominal defocus min: 1.46 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Liquid nitrogen cooled / Specimen holder model: GATAN LIQUID NITROGEN
DateAug 17, 2015
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 31 / Average electron dose: 18 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each micrograph
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: OTHER / Software - Name: CTFFIND3, EMAN2, and, AUTO3DEM / Number images used: 564
DetailsParticles were selected using boxer in Eman2. Random model generated using Auto3dem. Reconstruction done using Auto3dem.
FSC plot (resolution estimation)

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