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

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Entry
Database: EMDB / ID: EMD-5886
TitleElectron cryo-microscopy of nanobody AB6 in complex with poliovirus P1/Mahoney
Map data
Samplenanobody AB6 in complex with poliovirus P1/Mahoney:
virus / nanobody PVSP6A
Keywordspicornavirus / nanobody / antibody / VHH / poliovirus / mechanism of neutralization
Function / homology
Function and homology information


suppression by virus of host translation initiation factor activity / suppression by virus of host MDA-5 activity / suppression by virus of host RIG-I activity / picornain 2A / pore-mediated entry of viral genome into host cell / suppression by virus of host mRNA export from nucleus / suppression by virus of host MAVS activity / picornain 3C / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane ...suppression by virus of host translation initiation factor activity / suppression by virus of host MDA-5 activity / suppression by virus of host RIG-I activity / picornain 2A / pore-mediated entry of viral genome into host cell / suppression by virus of host mRNA export from nucleus / suppression by virus of host MAVS activity / picornain 3C / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / RNA-protein covalent cross-linking / positive stranded viral RNA replication / integral to membrane of host cell / pore formation by virus in membrane of host cell / virion assembly / viral capsid / protein complex oligomerization / endocytosis involved in viral entry into host cell / nucleoside-triphosphate phosphatase / ion channel activity / induction by virus of host autophagy / RNA-directed RNA polymerase / cysteine-type endopeptidase activity / viral RNA genome replication / RNA-directed 5'-3' RNA polymerase activity / RNA helicase activity / transcription, DNA-templated / virion attachment to host cell / host cell nucleus / structural molecule activity / RNA binding / membrane / ATP binding / metal ion binding
Similarity search - Function
Poliovirus 3A protein-like / Poliovirus 3A protein like / Poliovirus core protein 3a, soluble domain / Picornavirus core protein 2A / Picornavirus 2B protein / Picornavirus 2B protein / Peptidase C3, 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 / Poliovirus core protein 3a, soluble domain / Picornavirus core protein 2A / Picornavirus 2B protein / Picornavirus 2B protein / Peptidase C3, 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 / RNA-directed RNA polymerase, catalytic domain / RdRp of positive ssRNA viruses catalytic domain profile. / Reverse transcriptase/Diguanylate cyclase domain / 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 speciesHuman poliovirus 1 / Camelus dromedarius (Arabian camel)
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
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jbd
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3jbd
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

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)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.68 Å/pix.
x 320 pix.
= 537.92 Å
1.68 Å/pix.
x 320 pix.
= 537.92 Å
1.68 Å/pix.
x 320 pix.
= 537.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

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 / Details: 1 / Number of components: 2
Oligomeric State: 60 nanobody VHH monomers bind to each poliovirion
MassTheoretical: 9.0 MDa / Experimental: 9.0 MDa / Measured by: 1

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Component #1: virus, Human poliovirus 1

VirusName: Human poliovirus 1 / Class: VIRION / Enveloped: No / Empty: No / Isolate: SEROTYPE
MassTheoretical: 9 MDa / Experimental: 9 MDa
SpeciesSpecies: Human poliovirus 1 / Serotype: Mahoney
Source (natural)Host Species: Homo sapiens (human) / Host category: VERTEBRATES

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Component #2: protein, nanobody PVSP6A

ProteinName: nanobody PVSP6A / a.k.a: nanobody AB6
Details: Each virus is decorated with 60 copies of nanobody PVSP6A.
Recombinant expression: Yes / Number of Copies: 60
SourceSpecies: Camelus dromedarius (Arabian camel)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pHEN6(c) / Strain: WK6

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 1 mg/mL / Buffer solution: 145 mM NaCl, 50 mM Na2HPO4.12H2O / pH: 7.4
Support filmQuantifoil R2/2
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 154 K / Method: 2 second blot

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Oct 17, 2012
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 25 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 96000 X (nominal), 89232 X (calibrated) / Cs: 2.6 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000 - 3000 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN ULTRASCAN 4000 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 6223 / Sampling size: 15 µm / Bit depth: 16

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: I (正20面体型対称) / Number of projections: 57282 / Details: The particles were processed using Frealign.
3D reconstructionSoftware: Frealign / CTF correction: per particle / Resolution: 4.8 Å / Resolution method: FSC 0.143, semi-independent

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Atomic model buiding

Modeling #1Software: REFMAC5, COOT, SPDBV / Refinement protocol: rigid body
Target criteria: Stereochemically restrained maximum likelihood refinement of both Fourier phase and amplitude agreement
Refinement space: RECIPROCAL
Details: Rigid 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.
Input PDB model: 1HXS
Modeling #2Software: REFMAC5, COOT, SPDBV / Refinement protocol: rigid body
Target criteria: Stereochemically restrained maximum likelihood refinement of both Fourier phase and amplitude agreement
Refinement space: RECIPROCAL
Details: Rigid 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.
Input PDB model: 2PLV
Modeling #3Software: REFMAC5, COOT, SPDBV / Refinement protocol: rigid body
Target criteria: Stereochemically restrained maximum likelihood refinement of both Fourier phase and amplitude agreement
Refinement space: RECIPROCAL
Details: Rigid 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.
Input PDB model: 4BEL
Modeling #4Software: REFMAC5, COOT, SPDBV / Refinement protocol: rigid body
Target criteria: Stereochemically restrained maximum likelihood refinement of both Fourier phase and amplitude agreement
Refinement space: RECIPROCAL
Details: Rigid 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.
Input PDB model: 2X1P
Output model

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