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- PDB-5ktz: expanded poliovirus in complex with VHH 12B -

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Basic information

Entry
Database: PDB / ID: 5ktz
Titleexpanded poliovirus in complex with VHH 12B
Components
  • Genome polyprotein
  • VHH 12B
  • VP1
  • VP2
KeywordsVIRUS/immune system / poliovirus / VHH / nanobody / 80S / expanded / single domain antibody / VIRUS-immune system complex
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 / T=pseudo3 icosahedral viral capsid / picornain 3C / 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
Specimen sourcePoliovirus type 1
Camelus dromedarius (Arabian camel)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 4.3 Å resolution
AuthorsStrauss, M. / Schotte, L. / Filman, D.J. / Hogle, J.M.
CitationJournal: J. Virol. / Year: 2017
Title: Cryo-electron Microscopy Structures of Expanded Poliovirus with VHHs Sample the Conformational Repertoire of the Expanded State.
Authors: Mike Strauss / Lise Schotte / Krishanthi S Karunatilaka / David J Filman / James M Hogle
Abstract: By using cryo-electron microscopy, expanded 80S-like poliovirus virions (poliovirions) were visualized in complexes with four 80S-specific camelid VHHs (Nanobodies). In all four complexes, the VHHs ...By using cryo-electron microscopy, expanded 80S-like poliovirus virions (poliovirions) were visualized in complexes with four 80S-specific camelid VHHs (Nanobodies). In all four complexes, the VHHs bind to a site on the top surface of the capsid protein VP3, which is hidden in the native virus. Interestingly, although the four VHHs bind to the same site, the structures of the expanded virus differ in detail in each complex, suggesting that each of the Nanobodies has sampled a range of low-energy structures available to the expanded virion. By stabilizing unique structures of expanded virions, VHH binding permitted a more detailed view of the virus structure than was previously possible, leading to a better understanding of the expansion process that is a critical step in infection. It is now clear which polypeptide chains become disordered and which become rearranged. The higher resolution of these structures also revealed well-ordered conformations for the EF loop of VP2, the GH loop of VP3, and the N-terminal extensions of VP1 and VP2, which, in retrospect, were present in lower-resolution structures but not recognized. These structural observations help to explain preexisting mutational data and provide insights into several other stages of the poliovirus life cycle, including the mechanism of receptor-triggered virus expansion.
IMPORTANCE: When poliovirus infects a cell, it undergoes a change in its structure in order to pass RNA through its protein coat, but this altered state is short-lived and thus poorly understood. The structures of poliovirus bound to single-domain antibodies presented here capture the altered virus in what appear to be intermediate states. A careful analysis of these structures lets us better understand the molecular mechanism of infection and how these changes in the virus lead to productive-infection events.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jul 12, 2016 / Release: Nov 2, 2016
RevisionDateData content typeGroupCategoryItemProviderType
1.0Nov 2, 2016Structure modelrepositoryInitial release
1.1Nov 30, 2016Structure modelDatabase references
1.2Feb 1, 2017Structure modelDatabase references / Other
1.3Feb 8, 2017Structure modelDatabase references
1.4Sep 27, 2017Structure modelAuthor supporting evidence / Data collectionem_image_scans / em_software / pdbx_audit_support_pdbx_audit_support.funding_organization
1.5Jul 18, 2018Structure modelData collection / Experimental preparationem_sample_support / em_software_em_sample_support.grid_type / _em_software.image_processing_id / _em_software.name

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Structure visualization

Movie
  • Biological unit as complete icosahedral assembly
  • Imaged by Jmol
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  • Biological unit as icosahedral pentamer
  • Imaged by Jmol
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  • Biological unit as icosahedral 23 hexamer
  • Imaged by Jmol
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  • Deposited structure unit
  • Imaged by Jmol
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  • Simplified surface model + fitted atomic model
  • EMDB-8284
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  • Superimposition on EM map
  • EMDB-8284
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
1: VP1
2: VP2
3: Genome polyprotein
7: VHH 12B


Theoretical massNumber of molelcules
Total (without water)93,8194
Polyers93,8194
Non-polymers00
Water0
1
1: VP1
2: VP2
3: Genome polyprotein
7: VHH 12B
x 60


Theoretical massNumber of molelcules
Total (without water)5,629,131240
Polyers5,629,131240
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation60
2


  • idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
3
1: VP1
2: VP2
3: Genome polyprotein
7: VHH 12B
x 5


  • icosahedral pentamer
  • 469 kDa, 20 polymers
Theoretical massNumber of molelcules
Total (without water)469,09420
Polyers469,09420
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation5
4
1: VP1
2: VP2
3: Genome polyprotein
7: VHH 12B
x 6


  • icosahedral 23 hexamer
  • 563 kDa, 24 polymers
Theoretical massNumber of molelcules
Total (without water)562,91324
Polyers562,91324
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation6
5


  • idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1

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Components

#1: Protein/peptide VP1


Mass: 25291.594 Da / Num. of mol.: 1 / Fragment: UNP residues 636-858 / Source: (gene. exp.) Poliovirus type 1 (strain Mahoney) / Strain: Mahoney / Production host: Homo sapiens (human) / References: UniProt: P03300
#2: Protein/peptide VP2


Mass: 29677.301 Da / Num. of mol.: 1 / Fragment: UNP residues 70-338 / Source: (gene. exp.) Poliovirus type 1 (strain Mahoney) / Strain: Mahoney / Production host: Homo sapiens (human) / References: UniProt: P03300
#3: Protein/peptide Genome polyprotein


Mass: 25777.613 Da / Num. of mol.: 1 / Fragment: UNP residues 342-572 / Source: (gene. exp.) Poliovirus type 1 / Strain: Mahoney / Production host: Homo sapiens (human) / References: UniProt: P03300
#4: Protein/peptide VHH 12B


Mass: 13072.334 Da / Num. of mol.: 1 / Source: (gene. exp.) Camelus dromedarius (Arabian camel) / Production host: Escherichia coli K-12 (bacteria)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: expanded poliovirus in complex with VHH 12B / Type: COMPLEX / Entity ID: 1, 2, 3, 4 / Source: MULTIPLE SOURCES
Molecular weightValue: 9 MDa / Experimental value: NO
Source (natural)Organism: Poliovirus type 1 (strain Mahoney)
Buffer solutionpH: 7
SpecimenConc.: 0.45 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 30 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0151 / Classification: refinement
EM software
IDNameVersionCategory
1e2boxer.pyparticle selection
2SerialEMimage acquisition
4CTFFIND3CTF correction
7Cootmodel fitting
8Cootother
9RELION1.3initial Euler assignment
10FREALIGN9final Euler assignment
11Relion 1.3classification
12GeFrealign3D reconstruction
13REFMAC 5.8.01245model refinement
14FREALIGN9.093D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: I
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 17627 / Symmetry type: POINT
Atomic model buildingDetails: Fitting protocol: rigid body restraint of structurally conserved areas, and stereochemically restrained, icosahedrally restrained flexible fitting of areas that would otherwise disagree with the map. Refinement space: reciprocal, using both Fourier amplitudes and phases.
Ref protocol: OTHER / Ref space: RECIPROCAL
RefineCorrelation coeff Fo to Fc: 0.733 / Overall SU B: 38.562 / Overall SU ML: 0.589 / Overall ESU R: 1.228
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Solvent computationSolvent model details: PARAMETERS FOR MASK CACLULATION
Displacement parametersB iso mean: 24.691 Å2 / Aniso B11: 0.06 Å2 / Aniso B12: -0.82 Å2 / Aniso B13: -0.07 Å2 / Aniso B22: -0.01 Å2 / Aniso B23: -0.43 Å2 / Aniso B33: -0.05 Å2
Least-squares processR factor R work: 0.47088 / R factor obs: 0.47088 / Highest resolution: 4.3 Å / Lowest resolution: 98.6 Å / Number reflection obs: 235686 / Percent reflection obs: 99.99
Refine LS restraints
Refine IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0100.01942314
ELECTRON MICROSCOPYr_bond_other_d
ELECTRON MICROSCOPYr_angle_refined_deg2.0471.94657698
ELECTRON MICROSCOPYr_angle_other_deg
ELECTRON MICROSCOPYr_dihedral_angle_1_deg0.2735.0005220
ELECTRON MICROSCOPYr_dihedral_angle_2_deg5.02523.4621814
ELECTRON MICROSCOPYr_dihedral_angle_3_deg2.14815.0006554
ELECTRON MICROSCOPYr_dihedral_angle_4_deg1.38815.000242
ELECTRON MICROSCOPYr_chiral_restr0.1510.2006456
ELECTRON MICROSCOPYr_gen_planes_refined0.0010.02132266
ELECTRON MICROSCOPYr_gen_planes_other
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it
ELECTRON MICROSCOPYr_mcbond_other
ELECTRON MICROSCOPYr_mcangle_it
ELECTRON MICROSCOPYr_mcangle_other
ELECTRON MICROSCOPYr_scbond_it
ELECTRON MICROSCOPYr_scbond_other
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other
ELECTRON MICROSCOPYr_long_range_B_refined
ELECTRON MICROSCOPYr_long_range_B_other
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS shellHighest resolution: 4.3 Å / R factor R work: 0.539 / Lowest resolution: 4.532 Å / Number reflection R free: 0 / Number reflection R work: 34480 / Total number of bins used: 10 / Percent reflection obs: 99.97

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