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- PDB-8ctg: Extracellular architecture of an engineered canonical Wnt signali... -

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Basic information

Entry
Database: PDB / ID: 8ctg
TitleExtracellular architecture of an engineered canonical Wnt signaling ternary complex
Components
  • Frizzled-8
  • Low-density lipoprotein receptor-related protein 6
  • Protein Wnt-8
KeywordsSIGNALING PROTEIN / signaling complex / beta-catenin
Function / homology
Function and homology information


negative regulation of cardiac cell fate specification / Spemann organizer formation / neural crest cell fate commitment / Wnt signaling pathway involved in somitogenesis / Wnt-Frizzled-LRP5/6 complex / Regulation of FZD by ubiquitination / Negative regulation of TCF-dependent signaling by WNT ligand antagonists / Asymmetric localization of PCP proteins / Signaling by RNF43 mutants / neural crest formation ...negative regulation of cardiac cell fate specification / Spemann organizer formation / neural crest cell fate commitment / Wnt signaling pathway involved in somitogenesis / Wnt-Frizzled-LRP5/6 complex / Regulation of FZD by ubiquitination / Negative regulation of TCF-dependent signaling by WNT ligand antagonists / Asymmetric localization of PCP proteins / Signaling by RNF43 mutants / neural crest formation / receptor-mediated endocytosis involved in cholesterol transport / embryonic axis specification / non-canonical Wnt signaling pathway / kinase inhibitor activity / Wnt receptor activity / low-density lipoprotein particle receptor activity / toxin transmembrane transporter activity / negative regulation of smooth muscle cell apoptotic process / Wnt-protein binding / cellular response to cholesterol / midbrain dopaminergic neuron differentiation / negative regulation of protein serine/threonine kinase activity / dopaminergic neuron differentiation / frizzled binding / neural crest cell differentiation / Wnt signalosome / Disassembly of the destruction complex and recruitment of AXIN to the membrane / anterior/posterior axis specification / neuronal dense core vesicle / canonical Wnt signaling pathway / coreceptor activity / positive regulation of cell cycle / Regulation of FZD by ubiquitination / TCF dependent signaling in response to WNT / PDZ domain binding / G protein-coupled receptor activity / protein localization to plasma membrane / cell-cell adhesion / response to peptide hormone / Wnt signaling pathway / positive regulation of DNA-binding transcription factor activity / T cell differentiation in thymus / positive regulation of cytosolic calcium ion concentration / chemical synaptic transmission / cytoplasmic vesicle / early endosome membrane / collagen-containing extracellular matrix / angiogenesis / protease binding / positive regulation of protein phosphorylation / membrane raft / signaling receptor binding / neuronal cell body / synapse / ubiquitin protein ligase binding / positive regulation of DNA-templated transcription / Golgi apparatus / cell surface / endoplasmic reticulum / protein homodimerization activity / positive regulation of transcription by RNA polymerase II / extracellular region / identical protein binding / plasma membrane
Similarity search - Function
Wnt-8 protein / Protein Wnt-8A/8C / Frizzled 8, cysteine-rich domain / Wnt protein, conserved site / Wnt-1 family signature. / Wnt / Wnt, C-terminal domain / wnt family / found in Wnt-1 / Low density lipoprotein receptor-related protein 5/6 ...Wnt-8 protein / Protein Wnt-8A/8C / Frizzled 8, cysteine-rich domain / Wnt protein, conserved site / Wnt-1 family signature. / Wnt / Wnt, C-terminal domain / wnt family / found in Wnt-1 / Low density lipoprotein receptor-related protein 5/6 / Frizzled/Smoothened, transmembrane domain / Frizzled/Smoothened family membrane region / Frizzled/Smoothened family membrane region / Frizzled/secreted frizzled-related protein / Frizzled / Frizzled domain / Frizzled cysteine-rich domain superfamily / Fz domain / Frizzled (fz) domain profile. / Low-density lipoprotein receptor repeat class B / LDL-receptor class B (LDLRB) repeat profile. / LDLR class B repeat / Low-density lipoprotein-receptor YWTD domain / Low-density lipoprotein receptor domain class A / Low-density lipoprotein (LDL) receptor class A, conserved site / LDL-receptor class A (LDLRA) domain signature. / LDL-receptor class A (LDLRA) domain profile. / Low-density lipoprotein receptor domain class A / Low-density lipoprotein (LDL) receptor class A repeat / LDL receptor-like superfamily / Six-bladed beta-propeller, TolB-like / Coagulation Factor Xa inhibitory site / GPCR, family 2-like / G-protein coupled receptors family 2 profile 2. / Epidermal growth factor-like domain. / EGF-like domain signature 2. / EGF-like domain
Similarity search - Domain/homology
PALMITOLEIC ACID / Low-density lipoprotein receptor-related protein 6 / Protein Wnt-8 / Frizzled-8
Similarity search - Component
Biological speciesMus musculus (house mouse)
Xenopus laevis (African clawed frog)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsTsutsumi, N. / Jude, K.M. / Garcia, K.C.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R37AI051321 United States
Howard Hughes Medical Institute (HHMI) United States
Ludwig Institute for Cancer Research (LICR) United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Structure of the Wnt-Frizzled-LRP6 initiation complex reveals the basis for coreceptor discrimination.
Authors: Naotaka Tsutsumi / Sunhee Hwang / Deepa Waghray / Simon Hansen / Kevin M Jude / Nan Wang / Yi Miao / Caleb R Glassman / Nathanael A Caveney / Claudia Y Janda / Rami N Hannoush / K Christopher Garcia /
Abstract: Wnt morphogens are critical for embryonic development and tissue regeneration. Canonical Wnts form ternary receptor complexes composed of tissue-specific Frizzled (Fzd) receptors together with the ...Wnt morphogens are critical for embryonic development and tissue regeneration. Canonical Wnts form ternary receptor complexes composed of tissue-specific Frizzled (Fzd) receptors together with the shared LRP5/6 coreceptors to initiate β-catenin signaling. The cryo-EM structure of a ternary initiation complex of an affinity-matured XWnt8-Frizzled8-LRP6 complex elucidates the basis of coreceptor discrimination by canonical Wnts by means of their N termini and linker domains that engage the LRP6 E1E2 domain funnels. Chimeric Wnts bearing modular linker "grafts" were able to transfer LRP6 domain specificity between different Wnts and enable non-canonical Wnt5a to signal through the canonical pathway. Synthetic peptides comprising the linker domain serve as Wnt-specific antagonists. The structure of the ternary complex provides a topological blueprint for the orientation and proximity of Frizzled and LRP6 within the Wnt cell surface signalosome.
History
DepositionMay 14, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 15, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 22, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Frizzled-8
B: Protein Wnt-8
C: Low-density lipoprotein receptor-related protein 6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)125,3774
Polymers125,1233
Non-polymers2541
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Frizzled-8 / / Fz-8 / mFz8


Mass: 15061.343 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Fzd8 / Production host: Drosophila melanogaster (fruit fly) / References: UniProt: Q61091
#2: Protein Protein Wnt-8 / XWnt-8


Mass: 36676.590 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: wnt8 / Production host: Drosophila melanogaster (fruit fly) / References: UniProt: P28026
#3: Protein Low-density lipoprotein receptor-related protein 6 / LRP-6


Mass: 73384.648 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LRP6 / Production host: Drosophila melanogaster (fruit fly) / References: UniProt: O75581
#4: Chemical ChemComp-PAM / PALMITOLEIC ACID / Palmitoleic acid


Mass: 254.408 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H30O2
Has ligand of interestN

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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
IDNameTypeDetailsEntity IDParent-IDSource
1haXWnt8-mFzd8CRD-hLRP6E1E2COMPLEXhigh-affinity XWnt8 variant in complex with mFzd8 CRD and hLRP6 E1E2#1-#30RECOMBINANT
2Frizzled-8COMPLEX#11RECOMBINANT
3Protein Wnt-8COMPLEX#21RECOMBINANT
4Low-density lipoprotein receptor-related protein 6COMPLEX#31RECOMBINANT
Molecular weightValue: 0.13 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
23Xenopus laevis (African clawed frog)8355
12Mus musculus (house mouse)10090
34Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Drosophila melanogaster (fruit fly)7227
23Drosophila melanogaster (fruit fly)7227
34Drosophila melanogaster (fruit fly)7227
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
120 mMHepes-sodium salt1
2150 mMsodium chloride1
32 mMcalcium chloride1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: haXWnt8-mFzd8CRD-hLRP6E1E2
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 3 s blotting before plunging

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 29000 X / Calibrated magnification: 58680 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 55 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10077

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4cryoSPARC3.3CTF correction
9PHENIXmodel refinement
10cryoSPARC3.3initial Euler assignment
11cryoSPARC3.3final Euler assignment
12cryoSPARC3.3classification
13cryoSPARC3.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3840364 / Details: Particles include duplicates and junk.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 82235
Details: Model resolution is substantially lower and estimated to be ~10 angstrom based on the map-model FSC.
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0045134
ELECTRON MICROSCOPYf_angle_d0.9617138
ELECTRON MICROSCOPYf_dihedral_angle_d7.5991181
ELECTRON MICROSCOPYf_chiral_restr0.047931
ELECTRON MICROSCOPYf_plane_restr0.0061040

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