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- PDB-9g6v: Dissociated FMDV SAT2 Pentamer in complex with ultralong Fab117 -

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Basic information

Entry
Database: PDB / ID: 9g6v
TitleDissociated FMDV SAT2 Pentamer in complex with ultralong Fab117
Components
  • (Genome polyprotein) x 3
  • Fab117 Heavy Chain
KeywordsVIRUS LIKE PARTICLE / disrupted / dissociated / VLP / internal epitope / pan-specific / ultralong
Function / homology
Function and homology information


symbiont-mediated perturbation of host chromatin organization / ribonucleoside triphosphate phosphatase activity / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / regulation of translation / channel activity / monoatomic ion transmembrane transport / clathrin-dependent endocytosis of virus by host cell / RNA helicase activity / viral protein processing ...symbiont-mediated perturbation of host chromatin organization / ribonucleoside triphosphate phosphatase activity / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / regulation of translation / channel activity / monoatomic ion transmembrane transport / clathrin-dependent endocytosis of virus by host cell / RNA helicase activity / viral protein processing / host cell endoplasmic reticulum membrane / cysteine-type endopeptidase activity / viral RNA genome replication / RNA-directed RNA polymerase activity / DNA-templated transcription / virion attachment to host cell / host cell nucleus / structural molecule activity / proteolysis / RNA binding / ATP binding / membrane
Similarity search - Function
Peptidase C28, foot-and-mouth virus L-proteinase / Foot-and-mouth virus L-proteinase / Aphthovirus leader protease (L(pro)) domain profile. / Foot-and-mouth disease virus VP1 coat / Capsid protein VP4, Picornavirus / Viral protein VP4 subunit / Capsid protein VP4 superfamily, Picornavirus / Helicase/polymerase/peptidase polyprotein, Calicivirus-type / Picornavirus coat protein / Papain-like cysteine peptidase superfamily ...Peptidase C28, foot-and-mouth virus L-proteinase / Foot-and-mouth virus L-proteinase / Aphthovirus leader protease (L(pro)) domain profile. / Foot-and-mouth disease virus VP1 coat / Capsid protein VP4, Picornavirus / Viral protein VP4 subunit / Capsid protein VP4 superfamily, Picornavirus / Helicase/polymerase/peptidase polyprotein, Calicivirus-type / Picornavirus coat protein / Papain-like cysteine peptidase superfamily / Peptidase C3A/C3B, picornaviral / 3C cysteine protease (picornain 3C) / Picornavirales 3C/3C-like protease domain / Picornavirales 3C/3C-like protease domain profile. / 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
Biological speciesFoot-and-mouth disease virus SAT 2
Bos taurus (domestic cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsClarke, J.D. / Duyvesteyn, H.M.E. / Ren, J. / Fry, E.E. / Owens, R.J. / Stuart, D.I. / Hammond, J.A.
Funding support United Kingdom, 4items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/M011224/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/N00065X/1 United Kingdom
Wellcome Trust060208/Z/00/Z United Kingdom
Wellcome Trust203141/Z/16/Z United Kingdom
CitationJournal: J Gen Virol / Year: 2024
Title: A broadly reactive ultralong bovine antibody that can determine the integrity of foot-and-mouth disease virus capsids.
Authors: John D Clarke / Helen M E Duyvesteyn / Eva Perez-Martin / Undīne Latišenko / Claudine Porta / Kathleen V Humphreys / Abigail L Hay / Jingshan Ren / Elizabeth E Fry / Erwin van den Born / ...Authors: John D Clarke / Helen M E Duyvesteyn / Eva Perez-Martin / Undīne Latišenko / Claudine Porta / Kathleen V Humphreys / Abigail L Hay / Jingshan Ren / Elizabeth E Fry / Erwin van den Born / Bryan Charleston / Marie Bonnet-Di Placido / Raymond J Owens / David I Stuart / John A Hammond /
Abstract: Foot-and-mouth disease vaccination using inactivated virus is suboptimal, as the icosahedral viral capsids often disassemble into antigenically distinct pentameric units during long-term storage, or ...Foot-and-mouth disease vaccination using inactivated virus is suboptimal, as the icosahedral viral capsids often disassemble into antigenically distinct pentameric units during long-term storage, or exposure to elevated temperature or lowered pH, and thus raise a response that is no longer protective. Furthermore, as foot-and-mouth disease virus (FMDV)'s seven serotypes are antigenically diverse, cross-protection from a single serotype vaccine is limited, and most existing mouse and bovine antibodies and camelid single-domain heavy chain-only antibodies are serotype-specific. For quality control purposes, there is a real need for pan-serotype antibodies that clearly distinguish between pentamer (12S) and protective intact FMDV capsid. To date, few cross-serotype bovine-derived antibodies have been reported in the literature. We identify a bovine antibody with an ultralong CDR-H3, Ab117, whose structural analysis reveals that it binds to a deep, hydrophobic pocket on the interior surface of the capsid via the CDR-H3. Main-chain and hydrophobic interactions provide broad serotype specificity. ELISA analysis confirms that Ab117 is a novel pan-serotype and conformational epitope-specific 12S reagent, suitable for assessing capsid integrity.
History
DepositionJul 19, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 30, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update
Revision 1.2Nov 20, 2024Group: Data collection / Derived calculations / Other
Category: em_admin / pdbx_database_status ...em_admin / pdbx_database_status / pdbx_struct_sheet_hbond / struct_conf / struct_sheet / struct_sheet_order / struct_sheet_range
Item: _em_admin.last_update / _pdbx_database_status.pdb_format_compatible

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
1: Genome polyprotein
2: Genome polyprotein
3: Genome polyprotein
B: Genome polyprotein
D: Genome polyprotein
F: Genome polyprotein
H: Genome polyprotein
J: Genome polyprotein
L: Genome polyprotein
N: Genome polyprotein
P: Genome polyprotein
A: Fab117 Heavy Chain
E: Fab117 Heavy Chain
I: Fab117 Heavy Chain
M: Fab117 Heavy Chain
Q: Fab117 Heavy Chain
C: Genome polyprotein
G: Genome polyprotein
K: Genome polyprotein
O: Genome polyprotein


Theoretical massNumber of molelcules
Total (without water)528,29820
Polymers528,29820
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Genome polyprotein


Mass: 24160.275 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Foot-and-mouth disease virus SAT 2 / Strain: SAT2/Zim/7/83 / Cell (production host): Epithelial-like / Cell line (production host): HEK293T / Organ (production host): Kidney / Production host: Homo sapiens (human) / References: UniProt: Q719N0
#2: Protein
Genome polyprotein


Mass: 24631.531 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Foot-and-mouth disease virus SAT 2 / Strain: SAT2/Zim/7/83 / Cell (production host): Epithelial-like / Cell line (production host): HEK293T / Organ (production host): Kidney / Production host: Homo sapiens (human) / References: UniProt: Q719N0
#3: Protein
Genome polyprotein


Mass: 24620.328 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Foot-and-mouth disease virus SAT 2 / Strain: SAT2/Zim/7/83 / Cell (production host): Epithelial-like / Cell line (production host): HEK293T / Organ (production host): Kidney / Production host: Homo sapiens (human) / References: UniProt: Q719N0
#4: Antibody
Fab117 Heavy Chain


Mass: 32247.385 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (domestic cattle) / Cell: B-Lymphocyte / Plasmid: pOPINBOVH / Cell line (production host): Expi293 / Organ (production host): Kidney / Production host: Homo sapiens (human)
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Complex of pentamer from Foot and Mouth Disease virus serotype SAT2 VLPs with Fab117.COMPLEXall0RECOMBINANT
2Pentamer from Foot and Mouth Disease Virus serotype SAT2 VLPsCOMPLEX#1-#31RECOMBINANT
3Fab117COMPLEX#41RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
31NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrainTissue
21Bos taurus (domestic cattle)9913
32Foot-and-mouth disease virus SAT 235292SAT2/ZIM/7/83
43Bos taurus (domestic cattle)9913B-Lymphocyte
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainCellPlasmid
21Homo sapiens (human)9606
32Homo sapiens (human)9606CRL-3216HEK293TVaccinia Virus
43Homo sapiens (human)9606Expi293pOPINBOVH
Buffer solutionpH: 7.55 / Details: 150 mM NaCl, 150 mM Tris-HCl, 40 mM Na3PO4
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium ChlorideNaCl1
2150 mMTrizma-HydrochlorideNH2C(CH2OH)3HCl1
340 mMSodium PhosphateNa3PO41
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Foot and mouth disease virus SAT2 serotype virus like particle pentamer unit incubated with Fab117.
Specimen supportDetails: Grids glow discharged using Harrick Plasma PDC-002-CE on 'High' setting.
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 4 K / Details: Vitrification carried out in air atmosphere.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2800 nm / Nominal defocus min: 1400 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4.4 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10965

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
7UCSF Chimeramodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 267890
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 110210 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00322445
ELECTRON MICROSCOPYf_angle_d0.41530585
ELECTRON MICROSCOPYf_dihedral_angle_d8.5937890
ELECTRON MICROSCOPYf_chiral_restr0.043290
ELECTRON MICROSCOPYf_plane_restr0.0033970

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