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- EMDB-5888: Electron cryo-microscopy of nanobody AB29 in complex with poliovi... -

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Entry
Database: EMDB / ID: 5888
TitleElectron cryo-microscopy of nanobody AB29 in complex with poliovirus P1/Mahoney
Map datareconstruction of nanobody AB29 in complex with poliovirus P1/Mahoney
Samplenanobody AB29 in complex with poliovirus P1/Mahoney:
virus / nanobody PVSP29F
Keywordspicornavirus / nanobody / antibody / VHH / poliovirus / mechanism of neutralization
Function / homologyPoliovirus 3A protein-like / Viral coat protein subunit / Peptidase C3A/C3B, picornaviral / Picornavirus/Calicivirus coat protein / Helicase, superfamily 3, single-stranded DNA/RNA virus / Poliovirus core protein 3a, soluble domain / P-loop containing nucleoside triphosphate hydrolase / RNA-directed RNA polymerase, C-terminal domain / Picornavirus capsid / picornavirus capsid protein ...Poliovirus 3A protein-like / Viral coat protein subunit / Peptidase C3A/C3B, picornaviral / Picornavirus/Calicivirus coat protein / Helicase, superfamily 3, single-stranded DNA/RNA virus / Poliovirus core protein 3a, soluble domain / P-loop containing nucleoside triphosphate hydrolase / RNA-directed RNA polymerase, C-terminal domain / Picornavirus capsid / picornavirus capsid protein / Picornavirus 2B protein / 3C cysteine protease (picornain 3C) / Picornavirus coat protein VP4 / RNA-directed RNA polymerase, catalytic domain / RNA dependent RNA polymerase / RNA helicase / Picornavirus core protein 2A / Picornavirus 2B protein / Picornavirus coat protein (VP4) / Poliovirus 3A protein like / RdRp of positive ssRNA viruses catalytic domain profile. / Peptidase S1, PA clan / Superfamily 3 helicase of positive ssRNA viruses domain profile. / Helicase, superfamily 3, single-stranded RNA virus / Peptidase C3, picornavirus core protein 2A / suppression by virus of host translation initiation factor activity / positive stranded viral RNA replication / 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 RIG-I activity / picornain 3C / T=pseudo3 icosahedral viral capsid / endocytosis involved in viral entry into host cell / RNA-protein covalent cross-linking / host cell cytoplasmic vesicle membrane / pore formation by virus in membrane of host cell / integral to membrane of host cell / RNA helicase activity / nucleoside-triphosphate phosphatase / virion assembly / viral capsid / RNA-directed RNA polymerase / induction by virus of host autophagy / ion channel activity / protein complex oligomerization / viral RNA genome replication / RNA-directed 5'-3' RNA polymerase activity / cysteine-type endopeptidase activity / transcription, DNA-templated / host cell nucleus / virion attachment to host cell / structural molecule activity / RNA binding / membrane / ATP binding / Genome polyprotein
Function and homology information
SourceHuman poliovirus 1 / Camelus dromedarius (Arabian camel)
Methodsingle particle reconstruction / cryo EM / 6.5 Å resolution
AuthorsSchotte L / Strauss M / Thys B / Halewyck H / Filman DJ / Bostina M / Hogle JM / Rombaut B
Citation
Journal: J. Virol. / Year: 2014
Title: Mechanism of action and capsid-stabilizing properties of VHHs with an in vitro antipolioviral activity.
Authors: Lise Schotte / Mike Strauss / Bert Thys / Hadewych Halewyck / David J Filman / Mihnea Bostina / James M Hogle / Bart Rombaut
Abstract: Previously, we reported on the in vitro antiviral activity of single-domain antibody fragments (VHHs) directed against poliovirus type 1. Five VHHs were found to neutralize poliovirus type 1 in an in ...Previously, we reported on the in vitro antiviral activity of single-domain antibody fragments (VHHs) directed against poliovirus type 1. Five VHHs were found to neutralize poliovirus type 1 in an in vitro setting and showed 50% effective concentrations (EC50s) in the nanomolar range. In the present study, we further investigated the mechanism of action of these VHHs. All five VHHs interfere at multiple levels of the viral replication cycle, as they interfere both with attachment of the virus to cells and with viral uncoating. The latter effect is consistent with their ability to stabilize the poliovirus capsid, as observed in a ThermoFluor thermal shift assay, in which the virus is gradually heated and the temperature causing 50% of the RNA to be released from the capsid is determined, either in the presence or in the absence of the VHHs. The VHH-capsid interactions were also seen to induce aggregation of the virus-VHH complexes. However, this observation cannot yet be linked to their mechanism of action. Cryo-electron microscopy (cryo-EM) reconstructions of two VHHs in complex with poliovirus type 1 show no conformational changes of the capsid to explain this aggregation. On the other hand, these reconstructions do show that the binding sites of VHHs PVSP6A and PVSP29F overlap the binding site for the poliovirus receptor (CD155/PVR) and span interfaces that are altered during receptor-induced conformational changes associated with cell entry. This may explain the interference at the level of cell attachment of the virus as well as their effect on uncoating.
Importance: The study describes the mechanism of neutralization and the capsid-stabilizing activity of five single-domain antibody fragments (VHHs) that have an in vitro neutralizing activity against poliovirus type 1. The results show that the VHHs interfere at multiple levels of the viral replication cycle (cell attachment and viral uncoating). These mechanisms are possibly shared by some conventional antibodies and may therefore provide some insight into the natural immune responses. Since the binding sites of two VHHs studied by cryo-EM are very similar to that of the receptor, the VHHs can be used as probes to study the authentic virus-cell interaction. The structures and conclusions in this study are original and raise interesting findings regarding virus-receptor interactions and the order of key events early in infection.
#1: Journal: 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 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.
Validation ReportPDB-ID: 3jbc

SummaryFull reportAbout validation report
DateDeposition: Jan 21, 2014 / Header (metadata) release: Feb 19, 2014 / Map release: Feb 19, 2014 / Last update: Jan 27, 2016

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-3jbc
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-3jbc
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_5888.map.gz (map file in CCP4 format, 128001 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
320 pix
1.68 Å/pix.
= 537.92 Å
320 pix
1.68 Å/pix.
= 537.92 Å
320 pix
1.68 Å/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 Level:0.0402 (by emdb), 0.07 (movie #1):
Minimum - Maximum-0.17195146 - 0.24653147
Average (Standard dev.)-0.00587815 (0.02206700)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions320320320
Origin-160-160-160
Limit159159159
Spacing320320320
CellA=B=C: 537.92 Å
α=β=γ: 90.0 deg.

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-0.1720.247-0.006

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Supplemental data

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Sample components

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Entire nanobody AB29 in complex with poliovirus P1/Mahoney

EntireName: nanobody AB29 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 PVSP29F

ProteinName: nanobody PVSP29F / a.k.a: nanobody AB29
Details: Each virus is decorated with 60 copies of nanobody PVSP29F.
Recombinant expression: Yes
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

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Oct 23, 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: 2.7 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: 1503 / Sampling size: 15 microns / Bit depth: 16

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: I (icosahedral) / Number of projections: 9764 / Details: The particles were processed using Frealign.
3D reconstructionSoftware: Frealign / CTF correction: per particle / Resolution: 6.5 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: 1QD0
Output model

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