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- EMDB-6435: VHH complexes with poliovirus: cryo-electron microscopy studies a... -

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Basic information

Entry
Database: EMDB / ID: EMD-6435
TitleVHH complexes with poliovirus: cryo-electron microscopy studies at near-atomic resolution
Map dataReconstruction of VHH-poliovirus (PVSS21E - Mahoney Type 1)
Sample
  • Sample: Nanobody PVSS21E in complex with poliovirus P1/Mahoney
  • Virus: Human poliovirus 1
  • Protein or peptide: PVSS21E
Keywordspoliovirus / nanobodies / VHH / neutralizing antibodies
Function / homology
Function and homology information


symbiont-mediated suppression of host translation initiation / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MDA-5 activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of RIG-I activity / picornain 2A / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / symbiont-mediated suppression of host mRNA export from nucleus / symbiont genome entry into host cell via pore formation in plasma membrane / ribonucleoside triphosphate phosphatase activity / picornain 3C / T=pseudo3 icosahedral viral capsid ...symbiont-mediated suppression of host translation initiation / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MDA-5 activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of RIG-I activity / picornain 2A / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / symbiont-mediated suppression of host mRNA export from nucleus / symbiont genome entry into host cell via pore formation in plasma membrane / ribonucleoside triphosphate phosphatase activity / picornain 3C / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / endocytosis involved in viral entry into host cell / : / nucleoside-triphosphate phosphatase / protein complex oligomerization / monoatomic ion channel activity / RNA helicase activity / induction by virus of host autophagy / 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 / host cell nucleus / structural molecule activity / virion attachment to host cell / proteolysis / RNA binding / ATP binding / membrane / metal ion binding
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. / 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
Biological speciesCamelus dromedarius (Arabian camel) / Human poliovirus 1
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsStrauss M / Schotte L / Thys B / Filman DJ / Hogle JM
CitationJournal: J Virol / Year: 2016
Title: Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site.
Authors: Mike Strauss / Lise Schotte / Bert Thys / David J Filman / James M Hogle /
Abstract: Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In ...Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion.
IMPORTANCE: We describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. All five ...IMPORTANCE: We describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. All five VHHs bind deep in the virus canyon at similar sites that overlap extensively with the binding site for the receptor (CD155). The binding surfaces on the VHHs are surprisingly extensive, but despite the use of similar binding surfaces on the virus, the binding surface on the VHHs is unique for each VHH. In four of the five complexes, the virus remains essentially unchanged, but for the fifth there are significant changes reminiscent of but smaller in magnitude than the changes associated with cell entry, suggesting that this VHH traps the virus in a previously undescribed early intermediate state. The neutralizing mechanisms of the VHHs and their potential use as quality control agents for the end game of poliovirus eradication are discussed.
History
DepositionAug 24, 2015-
Header (metadata) releaseSep 23, 2015-
Map releaseJan 27, 2016-
UpdateMar 30, 2016-
Current statusMar 30, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0088
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0088
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jbg
  • Surface level: 0.0088
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3jbg
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6435.gif

Downloads & links

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Map

FileDownload / File: emd_6435.map.gz / Format: CCP4 / Size: 500 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of VHH-poliovirus (PVSS21E - Mahoney Type 1)
Voxel sizeX=Y=Z: 0.985 Å
Density
Contour LevelBy AUTHOR: 0.0088 / Movie #1: 0.0088
Minimum - Maximum-0.02721315 - 0.05364892
Average (Standard dev.)-0.00113853 (±0.00435797)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-256-256-256
Dimensions512512512
Spacing512512512
CellA=B=C: 504.32 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.9850.9850.985
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z504.320504.320504.320
α/β/γ90.00090.00090.000
start NX/NY/NZ-34-31-64
NX/NY/NZ6963129
MAP C/R/S123
start NC/NR/NS-256-256-256
NC/NR/NS512512512
D min/max/mean-0.0270.054-0.001

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Supplemental data

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Sample components

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Entire : Nanobody PVSS21E in complex with poliovirus P1/Mahoney

EntireName: Nanobody PVSS21E in complex with poliovirus P1/Mahoney
Components
  • Sample: Nanobody PVSS21E in complex with poliovirus P1/Mahoney
  • Virus: Human poliovirus 1
  • Protein or peptide: PVSS21E

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Supramolecule #1000: Nanobody PVSS21E in complex with poliovirus P1/Mahoney

SupramoleculeName: Nanobody PVSS21E in complex with poliovirus P1/Mahoney
type: sample / ID: 1000 / Details: 1
Oligomeric state: 60 nanobody VHH monomers bind to each poliovirion
Number unique components: 2
Molecular weightExperimental: 9.0 MDa / Theoretical: 9.0 MDa / Method: 1

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Supramolecule #1: Human poliovirus 1

SupramoleculeName: Human poliovirus 1 / type: virus / ID: 1 / Name.synonym: poliovirus
Details: The viral capsid is decorated with 60 copies of a single-domain antibody (PVSS21E).
NCBI-ID: 12080 / Sci species name: Human poliovirus 1 / Database: NCBI / Virus type: VIRION / Virus isolate: SEROTYPE / Virus enveloped: No / Virus empty: No / Syn species name: poliovirus / Sci species serotype: 1
Host (natural)Organism: Homo sapiens (human) / synonym: VERTEBRATES
Molecular weightExperimental: 7 MDa / Theoretical: 7 MDa
Virus shellShell ID: 1 / Diameter: 350 Å / T number (triangulation number): 3

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Macromolecule #1: PVSS21E

MacromoleculeName: PVSS21E / type: protein_or_peptide / ID: 1 / Name.synonym: nanobody, VHH
Details: 60 copies of this single-domain antibody are attached to the viral capsid.
Number of copies: 60 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Camelus dromedarius (Arabian camel) / synonym: camel / Tissue: blood / Cell: lymocytes
Molecular weightExperimental: 15 KDa / Theoretical: 15 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: WK6 / Recombinant plasmid: pHEN6(c)

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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

Concentration1 mg/mL
BufferpH: 7.4 / Details: 145 mM NaCl, 50 mM Na2HPO4.12H2O
GridDetails: C-flat 1.2/1.3
VitrificationCryogen name: ETHANE / Chamber temperature: 154 K / Instrument: HOMEMADE PLUNGER / Method: 4 second blot

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 25380.7 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.26 mm / Nominal defocus max: -4.0 µm / Nominal defocus min: -1.4 µm / Nominal magnification: 23000
Sample stageSpecimen holder: Polara holder / Specimen holder model: OTHER
TemperatureMin: 80 K / Max: 110 K / Average: 80 K
Alignment procedureLegacy - Electron beam tilt params: 0
DetailsGatan K2 operated in Super-resolution mode
DateNov 27, 2013
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Digitization - Sampling interval: 2.5 µm / Number real images: 300 / Average electron dose: 25 e/Å2 / Bits/pixel: 8
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: per particle
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: OTHER / Software - Name: Frealign / Number images used: 24717
DetailsThe particles were processed using Frealign.

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Atomic model buiding 1

Initial modelPDB ID:

3qxu

SoftwareName: COOT, SPDBV, REFMAC5
DetailsUsing stereochemically and icosahedrally restrained maximum likelihood refinement in REFMAC5, a representative subset of the full atomic model with all neighbors present was built and refined to fit the corresponding subset of the experimental map. Portions of the model whose density resembled a structural homolog were identified and restrained to agree with the homolog. Detailed atomic models were constructed in areas of difference wherever the resolution of the map permitted. The Fourier-amplitude-weighted average cosine of the phase discrepancy was tracked.
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT
Target criteria: ML agreement with Fourier amplitudes and phases
Output model

PDB-3jbg:
Complex of poliovirus with VHH PVSS21E

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