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- EMDB-11106: Poliovirus type 3 (strain Saukett) stabilised virus-like particle... -

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Basic information

Entry
Database: EMDB / ID: EMD-11106
TitlePoliovirus type 3 (strain Saukett) stabilised virus-like particle (PV3 SC8) from a mammalian expression system.
Map dataMRC map filtered after local resolution in RELION. Recommended contour level 0.07 after inspection in Chimera.
Sample
  • Virus: Human poliovirus 3
    • Protein or peptide: Capsid proteins, VP1
    • Protein or peptide: Capsid proteins, VP0
    • Protein or peptide: Capsid proteins, VP3
  • Ligand: SPHINGOSINE
Function / homology
Function and homology information


caveolin-mediated endocytosis of virus by host cell / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of RIG-I activity / 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 MAVS activity / picornain 2A / symbiont-mediated suppression of host mRNA export from nucleus / ribonucleoside triphosphate phosphatase activity / symbiont genome entry into host cell via pore formation in plasma membrane / picornain 3C / T=pseudo3 icosahedral viral capsid ...caveolin-mediated endocytosis of virus by host cell / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of RIG-I activity / 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 MAVS activity / picornain 2A / symbiont-mediated suppression of host mRNA export from nucleus / ribonucleoside triphosphate phosphatase activity / symbiont genome entry into host cell via pore formation in plasma membrane / picornain 3C / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / viral capsid / : / nucleoside-triphosphate phosphatase / protein complex oligomerization / monoatomic ion channel activity / RNA helicase activity / DNA replication / 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 / virion attachment to host cell / structural molecule activity / 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
Genome polyprotein / Genome polyprotein
Similarity search - Component
Biological speciesHuman poliovirus 3
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsBahar MW / Porta C / Fry EE / Stuart DI
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Bill & Melinda Gates FoundationRG.IMCB.I8-TSA-083 United Kingdom
CitationJournal: NPJ Vaccines / Year: 2021
Title: Mammalian expression of virus-like particles as a proof of principle for next generation polio vaccines.
Authors: Mohammad W Bahar / Claudine Porta / Helen Fox / Andrew J Macadam / Elizabeth E Fry / David I Stuart /
Abstract: Global vaccination programs using live-attenuated oral and inactivated polio vaccine (OPV and IPV) have almost eradicated poliovirus (PV) but these vaccines or their production pose significant risk ...Global vaccination programs using live-attenuated oral and inactivated polio vaccine (OPV and IPV) have almost eradicated poliovirus (PV) but these vaccines or their production pose significant risk in a polio-free world. Recombinant PV virus-like particles (VLPs), lacking the viral genome, represent safe next-generation vaccines, however their production requires optimisation. Here we present an efficient mammalian expression strategy producing good yields of wild-type PV VLPs for all three serotypes and a thermostabilised variant for PV3. Whilst the wild-type VLPs were predominantly in the non-native C-antigenic form, the thermostabilised PV3 VLPs adopted the native D-antigenic conformation eliciting neutralising antibody titres equivalent to the current IPV and were indistinguishable from natural empty particles by cryo-electron microscopy with a similar stabilising lipidic pocket-factor in the VP1 β-barrel. This factor may not be available in alternative expression systems, which may require synthetic pocket-binding factors. VLPs equivalent to these mammalian expressed thermostabilized particles, represent safer non-infectious vaccine candidates for the post-eradication era.
History
DepositionMay 29, 2020-
Header (metadata) releaseDec 30, 2020-
Map releaseDec 30, 2020-
UpdateSep 21, 2022-
Current statusSep 21, 2022Processing site: PDBe / Status: Released

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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-6z6w
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6z6w
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11106.png

Downloads & links

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Map

FileDownload / File: emd_11106.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMRC map filtered after local resolution in RELION. Recommended contour level 0.07 after inspection in Chimera.
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.07 / Movie #1: 0.07
Minimum - Maximum-0.16487819 - 0.29022428
Average (Standard dev.)0.0041176737 (±0.022492874)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 378.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z378.000378.000378.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.1650.2900.004

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Supplemental data

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Sample components

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Entire : Human poliovirus 3

EntireName: Human poliovirus 3
Components
  • Virus: Human poliovirus 3
    • Protein or peptide: Capsid proteins, VP1
    • Protein or peptide: Capsid proteins, VP0
    • Protein or peptide: Capsid proteins, VP3
  • Ligand: SPHINGOSINE

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Supramolecule #1: Human poliovirus 3

SupramoleculeName: Human poliovirus 3 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: Recombinantly expressed virus-like particle of PV3 (strain Saukett).
NCBI-ID: 12086 / Sci species name: Human poliovirus 3 / Sci species strain: Saukett / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: SEROTYPE / Virus enveloped: No / Virus empty: Yes
Host (natural)Organism: Homo sapiens (human)
Host systemOrganism: mammal environmental sample (environmental samples)
Recombinant cell: BHK-21 / Recombinant plasmid: pMVA_PV3 SC8_PV-IRES
Molecular weightTheoretical: 5.85 MDa
Virus shellShell ID: 1 / Name: Virus shell 1 / Diameter: 310.0 Å / T number (triangulation number): 1

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Macromolecule #1: Capsid proteins, VP1

MacromoleculeName: Capsid proteins, VP1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human poliovirus 3
Molecular weightTheoretical: 33.562785 KDa
Recombinant expressionOrganism: mammal environmental sample (environmental samples)
SequenceString: GIEDLITEVA QGALTLSLPK QQDSLPDTKA SGPAHSKEVP ALTAVETGAT NPLVPSDTVQ TRHVIQRRSR SESTIESFFA RGACVAIIE VDNEEPTTRA QKLFAMWRIT YKDTVQLRRK LEFFTYSRFD MELTFVVTAN FTNTNNGHAL NQVYQIMYIP P GAPTPKSW ...String:
GIEDLITEVA QGALTLSLPK QQDSLPDTKA SGPAHSKEVP ALTAVETGAT NPLVPSDTVQ TRHVIQRRSR SESTIESFFA RGACVAIIE VDNEEPTTRA QKLFAMWRIT YKDTVQLRRK LEFFTYSRFD MELTFVVTAN FTNTNNGHAL NQVYQIMYIP P GAPTPKSW DDYTWQTSSN PSIFYTYGAA PARISVPYVG LANAYSHFYD GFAKVPLKTD ANDQIGDSLY SAMTVDDFGV LA IRVVNDH NPTKVTSKVR IYMKPKHVRV WCPRPPRAVP YYGPGVDYKD NLNPLSEKGL TTY

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Macromolecule #2: Capsid proteins, VP0

MacromoleculeName: Capsid proteins, VP0 / type: protein_or_peptide / ID: 2
Details: Sequence is given for the VP0 polypeptide. Mutations are numbered according to sequence numbering for mature polypeptides VP2 and VP4.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human poliovirus 3
Molecular weightTheoretical: 37.623023 KDa
Recombinant expressionOrganism: mammal environmental sample (environmental samples)
SequenceString: MGAQVSSQKV GAHENSNRAY GGSTINYTTI NYYKDSASNA ASKQDYSQDP SKFTEPLKDV LIKTAPALNS PNVEACGYSD RVLQLTIGN STITTQEAAN SVVAYGRWPE FIRDDEANPV DQPTEPDVAT CRFYTLDTVM WGKESKGWWW KLPDALRDMG L FGQNMYYH ...String:
MGAQVSSQKV GAHENSNRAY GGSTINYTTI NYYKDSASNA ASKQDYSQDP SKFTEPLKDV LIKTAPALNS PNVEACGYSD RVLQLTIGN STITTQEAAN SVVAYGRWPE FIRDDEANPV DQPTEPDVAT CRFYTLDTVM WGKESKGWWW KLPDALRDMG L FGQNMYYH YLGRSGYTVH VQCNASKFHQ GALGVFAIPE YCLAGDSDKQ RYTSYANANP GEKGGKFYSQ FNRDTAVTSP KR EFCPVDY LLGCGVLLGN AFVYPHQIIN LRTNNSATIV LPYVNAMAID SMVKHNNWGI AILPLSPLDF AQESSVEIPI TVT IAPMCS EFNGLRNVTA PKFQ

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Macromolecule #3: Capsid proteins, VP3

MacromoleculeName: Capsid proteins, VP3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human poliovirus 3
Molecular weightTheoretical: 26.3151 KDa
Recombinant expressionOrganism: mammal environmental sample (environmental samples)
SequenceString: GLPVLNTPGS NQYLTSDNYQ SPCAIPEFDV TPPIDIPGEV KNMMELAEID TMIPLNLENT KRNTMDMYRV TLSDSADLSQ PILCFSLSP ASDPRLSHTM LGEVLNYYTH WAGSLKFTFL FCGSMMATGK ILVAYAPPGA QPPTSRKEAM LGTHVIWDLG L QSSCTMVV ...String:
GLPVLNTPGS NQYLTSDNYQ SPCAIPEFDV TPPIDIPGEV KNMMELAEID TMIPLNLENT KRNTMDMYRV TLSDSADLSQ PILCFSLSP ASDPRLSHTM LGEVLNYYTH WAGSLKFTFL FCGSMMATGK ILVAYAPPGA QPPTSRKEAM LGTHVIWDLG L QSSCTMVV PWISNVTYRQ TTQDSFTEGG YISMFYQTRI VVPLSTPKSM SMLGFVSACN DFSVRLLRDT THISQSALPQ

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Macromolecule #4: SPHINGOSINE

MacromoleculeName: SPHINGOSINE / type: ligand / ID: 4 / Number of copies: 1 / Formula: SPH
Molecular weightTheoretical: 299.492 Da
Chemical component information

ChemComp-SPH:
SPHINGOSINE / Sphingosine

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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

Concentration1.0 mg/mL
BufferpH: 7
Component:
ConcentrationName
1.0 xDPBS
20.0 mMEDTAEthylenediaminetetraacetic acid

Details: 1 x DPBS, 20 mM EDTA, pH 7.0
GridModel: C-flat / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: LACEY / Support film - Film thickness: 3.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
Details: The specific type of grid used was Ultra-thin carbon support film, 3nm - on lacey carbon AGS187-4 from Agar Scientific.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: Double blotting with 4 ul of sample, followed by 4 second blot, before plunging..
DetailsRecombinantly expressed VLP of PV3.

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 37037 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 160000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-25 / Number grids imaged: 2 / Number real images: 1465 / Average exposure time: 5.0 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 14021
CTF correctionSoftware - Name: CTFFIND (ver. 4.0)
Software - details: CTFFIND4 in RELION was used for CTF correction
Startup modelType of model: EMDB MAP
EMDB ID:

3747


Details: Startup model was low pass filtered to 60 Angstroms.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 5 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: I (icosahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1)
Details: Final reconstruction was sharpened with Post-processing in RELION using an inverse B-factor of -82.6 Angstroms.
Number images used: 9630
DetailsPixels size was 1.35 A/pixel
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5o5b

DetailsInitial model was rigid body fitted using UCSF chimera, and the refined in real space using Phenix_real.space.refine.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient
Output model

PDB-6z6w:
Poliovirus type 3 (strain Saukett) stabilised virus-like particle (PV3 SC8) from a mammalian expression system.

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