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- EMDB-0056: Feline Calicivirus Strain F9 bound to a soluble ectodomain fragme... -

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Basic information

Entry
Database: EMDB / ID: EMD-0056
TitleFeline Calicivirus Strain F9 bound to a soluble ectodomain fragment of feline junctional adhesion molecule A - leading to assembly of a portal structure at a unique three-fold axis.
Map dataC3 reconstruction of Feline Calicivirus decorated with a soluble ectodomain fragment of the cellular receptor feline junctional adhesion molecule A.
Sample
  • Virus: Feline calicivirus strain F9
    • Virus: Feline calicivirus strain F9
      • Protein or peptide: Capsid proteinCapsid
    • Complex: feline junctional adhesion molecule A
      • Protein or peptide: Junctional adhesion molecule A
    • Complex: VP2 - portal.
      • Protein or peptide: VP2
  • Ligand: POTASSIUM IONPotassium
Function / homology
Function and homology information


establishment of endothelial intestinal barrier / regulation of membrane permeability / T=3 icosahedral viral capsid / intestinal absorption / bicellular tight junction / host cell cytoplasm / membrane => GO:0016020 / cell adhesion / plasma membrane
Similarity search - Function
Vesivirus VP2 / Vesivirus VP2 protein / Junctional adhesion molecule A / Calicivirus coat protein / Calicivirus coat protein / Immunoglobulin subtype 2 / Immunoglobulin C-2 Type / Immunoglobulin V-Type / Picornavirus/Calicivirus coat protein / Immunoglobulin V-set domain ...Vesivirus VP2 / Vesivirus VP2 protein / Junctional adhesion molecule A / Calicivirus coat protein / Calicivirus coat protein / Immunoglobulin subtype 2 / Immunoglobulin C-2 Type / Immunoglobulin V-Type / Picornavirus/Calicivirus coat protein / Immunoglobulin V-set domain / Viral coat protein subunit / Immunoglobulin V-set domain / Immunoglobulin subtype / Immunoglobulin / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Capsid protein / Protein VP2 / Junctional adhesion molecule A
Similarity search - Component
Biological speciesFelis catus (domestic cat) / Feline calicivirus strain F9 / FCV (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.75 Å
AuthorsConley MJ / Bhella D
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_UU_12014/7 United Kingdom
Biotechnology and Biological Sciences Research CouncilBB/J013854/1 United Kingdom
CitationJournal: Nature / Year: 2019
Title: Calicivirus VP2 forms a portal-like assembly following receptor engagement.
Authors: Michaela J Conley / Marion McElwee / Liyana Azmi / Mads Gabrielsen / Olwyn Byron / Ian G Goodfellow / David Bhella /
Abstract: To initiate infection, many viruses enter their host cells by triggering endocytosis following receptor engagement. However, the mechanisms by which non-enveloped viruses escape the endosome are ...To initiate infection, many viruses enter their host cells by triggering endocytosis following receptor engagement. However, the mechanisms by which non-enveloped viruses escape the endosome are poorly understood. Here we present near-atomic-resolution cryo-electron microscopy structures for feline calicivirus both undecorated and labelled with a soluble fragment of its cellular receptor, feline junctional adhesion molecule A. We show that VP2, a minor capsid protein encoded by all caliciviruses, forms a large portal-like assembly at a unique three-fold axis of symmetry, following receptor engagement. This assembly-which was not detected in undecorated virions-is formed of twelve copies of VP2, arranged with their hydrophobic N termini pointing away from the virion surface. Local rearrangement at the portal site leads to the opening of a pore in the capsid shell. We hypothesize that the portal-like assembly functions as a channel for the delivery of the calicivirus genome, through the endosomal membrane, into the cytoplasm of a host cell, thereby initiating infection. VP2 was previously known to be critical for the production of infectious virus; our findings provide insights into its structure and function that advance our understanding of the Caliciviridae.
History
DepositionJun 14, 2018-
Header (metadata) releaseAug 22, 2018-
Map releaseJan 16, 2019-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.05
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  • Surface view with fitted model
  • Atomic models: PDB-6gsi
  • Surface level: 0.05
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6gsi
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0056.png

Downloads & links

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Map

FileDownload / File: emd_0056.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationC3 reconstruction of Feline Calicivirus decorated with a soluble ectodomain fragment of the cellular receptor feline junctional adhesion molecule A.
Voxel sizeX=Y=Z: 1.065 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.11780139 - 0.21274377
Average (Standard dev.)0.00095921505 (±0.013303866)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-300-300-300
Dimensions600600600
Spacing600600600
CellA=B=C: 639.00006 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0651.0651.065
M x/y/z600600600
origin x/y/z0.0000.0000.000
length x/y/z639.000639.000639.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-300-300-300
NC/NR/NS600600600
D min/max/mean-0.1180.2130.001

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Supplemental data

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Sample components

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Entire : Feline calicivirus strain F9

EntireName: Feline calicivirus strain F9
Components
  • Virus: Feline calicivirus strain F9
    • Virus: Feline calicivirus strain F9
      • Protein or peptide: Capsid proteinCapsid
    • Complex: feline junctional adhesion molecule A
      • Protein or peptide: Junctional adhesion molecule A
    • Complex: VP2 - portal.
      • Protein or peptide: VP2
  • Ligand: POTASSIUM IONPotassium

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Supramolecule #1: Feline calicivirus strain F9

SupramoleculeName: Feline calicivirus strain F9 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: Virus was propagated in Crandell Reese Feline Kidney cells
Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Felis catus (domestic cat)
Virus shellShell ID: 1 / Name: Capsid / Diameter: 400.0 Å / T number (triangulation number): 3

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Supramolecule #2: Feline calicivirus strain F9

SupramoleculeName: Feline calicivirus strain F9 / type: virus / ID: 2 / Parent: 1 / Macromolecule list: #1 / Details: Icosahedral Capsid / NCBI-ID: 11981 / Sci species name: Feline calicivirus strain F9 / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Felis catus (domestic cat)
Virus shellShell ID: 2 / Name: Capsid / Diameter: 400.0 Å / T number (triangulation number): 3

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Supramolecule #3: feline junctional adhesion molecule A

SupramoleculeName: feline junctional adhesion molecule A / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Details: Soluble ectodomain fragment comprising Ig-like domains D1 and D2
Source (natural)Organism: Felis catus (domestic cat)
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster) / Recombinant cell: CHO cells

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Supramolecule #4: VP2 - portal.

SupramoleculeName: VP2 - portal. / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Details: Postulated to mediate endosome escape, this virion component is only present on the outer surface of the capsid following receptor engagement.
Source (natural)Organism: Feline calicivirus strain F9

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Macromolecule #1: Capsid protein

MacromoleculeName: Capsid protein / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: FCV (virus)
Molecular weightTheoretical: 73.346664 KDa
SequenceString: MCSTCANVLK YYNWDPHFRL VIDPNKFLSV GFCDNPLMCC YPELLPEFGT VWDCDQSPLQ IYLESILGDD EWASTYEAID PAVPPMHWD AAGKIFQPHP GVLMHHLIGE VAKAWDPNLP MFRLEADGDG SITAPEQGTP VGGVIAEPSA QMSAAADMAT G KSVDSEWE ...String:
MCSTCANVLK YYNWDPHFRL VIDPNKFLSV GFCDNPLMCC YPELLPEFGT VWDCDQSPLQ IYLESILGDD EWASTYEAID PAVPPMHWD AAGKIFQPHP GVLMHHLIGE VAKAWDPNLP MFRLEADGDG SITAPEQGTP VGGVIAEPSA QMSAAADMAT G KSVDSEWE AFFSFHTSVN WSTSETQGKI LFKQSLGPLL NPYLEHLAKL YVAWSGSIDV RFSISGSGVF GGKLAAIVVP PG VDPVQST SMLQYPHVLF DARQVEPVIF SIPDLRSTLY HLMSDTDTTS LVIMVYNDLI NPYANDSNSS GCIVTVETKP GAD FKFHLL KPPGSMLTHG SVPSDLIPKS SSLWIGNRHW TDITDFVIRP FVFQANRHFD FNQETAGWST PRYRPITITI SEKN GAKLG IGVATDYIVP GIPDGWPDTT IPEKLTPAGD YAITNKSGND ITTAAGYDGA DVIVNNTNFK GMYICGSLQR AWGDK KISN TAFITTATKV DNAIEPSNVI DMTKIAVYQD THVGKEVQTS DDTLSLLGYT GIGEQAIGSD RDRVVRISVL PETGAR GGN HPIFYKNSIK LGYVIRSIDV FNSQILHTSR QLSLNHYLLP PDSFAVYRII DSNGSWFDIG IDSDGFSFVG VSSIGKL EF PLTASYMGIQ LAKIRLASNI RSSMTKL

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Macromolecule #2: Junctional adhesion molecule A

MacromoleculeName: Junctional adhesion molecule A / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Felis catus (domestic cat)
Molecular weightTheoretical: 22.18065 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: AVYTSEPDVR VPEDKPAKLS CSYSGFSNPR VEWKFAHGDI TSLVCYKNKI TASYADRVTF SHSGITFHSV TRKDTGTYTC MVSDDGGNT YGEVSVQLTV LVPPSKPTVH IPSSATIGSR AVLTCSEKDG SPPSEYYWFK DGVRMPLEPK GNRAFSNSSY S LNEKTGEL ...String:
AVYTSEPDVR VPEDKPAKLS CSYSGFSNPR VEWKFAHGDI TSLVCYKNKI TASYADRVTF SHSGITFHSV TRKDTGTYTC MVSDDGGNT YGEVSVQLTV LVPPSKPTVH IPSSATIGSR AVLTCSEKDG SPPSEYYWFK DGVRMPLEPK GNRAFSNSSY S LNEKTGEL VFDPVSAWDT GEYTCEAQNG YGMPMRSEAV RMEA

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Macromolecule #3: VP2

MacromoleculeName: VP2 / type: protein_or_peptide / ID: 3 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: FCV (virus)
Molecular weightTheoretical: 12.209838 KDa
SequenceString:
MNSILGLIDT VTNTIGKAQQ IELDKAALGQ QRELALKRMK LDHQALNNQV EQFNKILEQR VQGPIQSVRL ARAAGFRVDP YSYTDQNFY DDQLNAIRLS YRNLFKN

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Macromolecule #4: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: K
Molecular weightTheoretical: 39.098 Da

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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: 7.2 / Details: Phosphate buffered saline
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsPurified virions were incubated in the presence of purified ectodomain fragments.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Number real images: 13865 / Average electron dose: 63.0 e/Å2
Details: Each micrograph was recorded as a movie of 50 individual fractions with a total dose of 63 e/angstrom squared
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 129884 / Details: Autopicking in Relion
CTF correctionSoftware - Name: Gctf / Details: CTF correction was implemented through Relion
Startup modelType of model: OTHER
Details: Previously published models calculated at lower resolution.
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 10 / Software - Name: RELION (ver. 2.1)
Details: Origin and orientation values were initially determined by 3D auto refine in RELION, assuming full icosahedral symmetry (i.e. with full gold-standard methods). Once orientations were ...Details: Origin and orientation values were initially determined by 3D auto refine in RELION, assuming full icosahedral symmetry (i.e. with full gold-standard methods). Once orientations were accurately determined relion_symmetry_expand was used to produce a STAR file with 60 orientations per particle. This was used for a focussed classification experiment in which a cylindrical mask was applied to a single three-fold axis for 3D classification without orientation refinement. One class showed the presence of the previously detected portal assembly. Particles contributed a median 3 views each. NOTE The reconstruction half-sets were calculated with C1 symmetry, however as three views per particle are present that are derived from icosahedral redundancy, C3 symmetry is imposed by the data.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.75 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1)
Details: Origins and orientations were originally assigned by 3D auto refinement with full icosahedral symmetry. Random group assignment was carried through the focussed classification process and ...Details: Origins and orientations were originally assigned by 3D auto refinement with full icosahedral symmetry. Random group assignment was carried through the focussed classification process and used to divide the data into two roughly even halves that had been initially refined independently.
Number images used: 58510
DetailsImages were motion-corrected using motioncor2 Defocus estimation was performed using GCTF
FSC plot (resolution estimation)

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