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- EMDB-5886: Electron cryo-microscopy of nanobody AB6 in complex with poliovir... -

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Basic information

Entry
Database: EMDB / ID: EMD-5886
TitleElectron cryo-microscopy of nanobody AB6 in complex with poliovirus P1/Mahoney
Map datareconstruction of nanobody AB6 in complex with poliovirus P1/Mahoney
Sample
  • Sample: nanobody AB6 in complex with poliovirus P1/Mahoney
  • Virus: Human poliovirus 1
  • Protein or peptide: nanobody PVSP6A
Keywordspicornavirus / nanobody / antibody / VHH / poliovirus / mechanism of neutralization
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 mRNA export from nucleus / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / symbiont genome entry into host cell via pore formation in plasma membrane / picornain 3C / ribonucleoside triphosphate phosphatase activity / 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 mRNA export from nucleus / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / symbiont genome entry into host cell via pore formation in plasma membrane / picornain 3C / ribonucleoside triphosphate phosphatase activity / 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 / DNA/RNA polymerase superfamily / Peptidase S1, PA clan / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Biological speciesCamelus dromedarius (Arabian camel) / Human poliovirus 1
Methodsingle particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsSchotte L / Strauss M / Thys B / Halewyck H / Filman DJ / Bostina M / Hogle JM / Rombaut B
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
DepositionJan 20, 2014-
Header (metadata) releaseFeb 19, 2014-
Map releaseFeb 19, 2014-
UpdateJan 27, 2016-
Current statusJan 27, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.04
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  • Surface view with fitted model
  • Atomic models: PDB-3jbd
  • Surface level: 0.04
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3jbd
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5886_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5886.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationreconstruction of nanobody AB6 in complex with poliovirus P1/Mahoney
Voxel sizeX=Y=Z: 1.681 Å
Density
Contour LevelBy EMDB: 0.0219 / Movie #1: 0.04
Minimum - Maximum-0.05857689 - 0.13004495
Average (Standard dev.)-0.00741233 (±0.01412011)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-160-160-160
Dimensions320320320
Spacing320320320
CellA=B=C: 537.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.6811.6811.681
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z537.920537.920537.920
α/β/γ90.00090.00090.000
start NX/NY/NZ-180-180-179
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS-160-160-160
NC/NR/NS320320320
D min/max/mean-0.0590.130-0.007

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Supplemental data

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Sample components

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Entire : nanobody AB6 in complex with poliovirus P1/Mahoney

EntireName: nanobody AB6 in complex with poliovirus P1/Mahoney
Components
  • Sample: nanobody AB6 in complex with poliovirus P1/Mahoney
  • Virus: Human poliovirus 1
  • Protein or peptide: nanobody PVSP6A

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Supramolecule #1000: nanobody AB6 in complex with poliovirus P1/Mahoney

SupramoleculeName: nanobody AB6 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 / NCBI-ID: 12080 / Sci species name: Human poliovirus 1 / Database: NCBI / Virus type: VIRION / Virus isolate: SEROTYPE / Virus enveloped: No / Virus empty: No / Sci species serotype: Mahoney
Host (natural)Organism: Homo sapiens (human) / synonym: VERTEBRATES
Molecular weightExperimental: 9 MDa / Theoretical: 9 MDa
Virus shellShell ID: 1 / Diameter: 350 Å / T number (triangulation number): 1

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Macromolecule #1: nanobody PVSP6A

MacromoleculeName: nanobody PVSP6A / type: protein_or_peptide / ID: 1 / Name.synonym: nanobody AB6
Details: Each virus is decorated with 60 copies of nanobody PVSP6A.
Number of copies: 60 / Recombinant expression: Yes
Source (natural)Organism: Camelus dromedarius (Arabian camel) / synonym: dromedary
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: Quantifoil R2/2
VitrificationCryogen name: ETHANE / Chamber temperature: 154 K / Instrument: HOMEMADE PLUNGER / Method: 2 second blot

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 89232 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.6 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 96000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DateOct 17, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 6223 / Average electron dose: 25 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: per particle
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.8 Å / Resolution method: OTHER / Software - Name: Frealign / Number images used: 57282
DetailsThe particles were processed using Frealign.

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Atomic model buiding 1

Initial modelPDB ID:

1hxs

SoftwareName: REFMAC5, COOT, SPDBV
DetailsRigid body with some flexible loops and side chains. LSQKAB was applied after each refinement cycle to re-impose exact icosahedral operators and rigid body constraints.
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT
Target criteria: Stereochemically restrained maximum likelihood refinement of both Fourier phase and amplitude agreement
Output model

PDB-3jbd:
Complex of poliovirus with VHH PVSP6A

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Atomic model buiding 2

Initial modelPDB ID:

2plv

SoftwareName: REFMAC5, COOT, SPDBV
DetailsRigid body with some flexible loops and side chains. LSQKAB was applied after each refinement cycle to re-impose exact icosahedral operators and rigid body constraints.
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT
Target criteria: Stereochemically restrained maximum likelihood refinement of both Fourier phase and amplitude agreement
Output model

PDB-3jbd:
Complex of poliovirus with VHH PVSP6A

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Atomic model buiding 3

Initial modelPDB ID:

4bel

SoftwareName: REFMAC5, COOT, SPDBV
DetailsRigid body with some flexible loops and side chains. LSQKAB was applied after each refinement cycle to re-impose exact icosahedral operators and rigid body constraints.
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT
Target criteria: Stereochemically restrained maximum likelihood refinement of both Fourier phase and amplitude agreement
Output model

PDB-3jbd:
Complex of poliovirus with VHH PVSP6A

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Atomic model buiding 4

Initial modelPDB ID:

2x1p

SoftwareName: REFMAC5, COOT, SPDBV
DetailsRigid body with some flexible loops and side chains. LSQKAB was applied after each refinement cycle to re-impose exact icosahedral operators and rigid body constraints.
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT
Target criteria: Stereochemically restrained maximum likelihood refinement of both Fourier phase and amplitude agreement
Output model

PDB-3jbd:
Complex of poliovirus with VHH PVSP6A

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