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- EMDB-26989: Extracellular architecture of an engineered canonical Wnt signali... -

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Basic information

Entry
Database: EMDB / ID: EMD-26989
TitleExtracellular architecture of an engineered canonical Wnt signaling ternary complex
Map datadeepEMhancer sharpened
Sample
  • Complex: haXWnt8-mFzd8CRD-hLRP6E1E2
    • Complex: Frizzled-8
      • Protein or peptide: Frizzled-8
    • Complex: Protein Wnt-8
      • Protein or peptide: Protein Wnt-8
    • Complex: Low-density lipoprotein receptor-related protein 6
      • Protein or peptide: Low-density lipoprotein receptor-related protein 6
  • Ligand: PALMITOLEIC ACID
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
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsTsutsumi N / Jude KM / Garcia KC
Funding support United States, 3 items
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, 2022-
Header (metadata) releaseMar 15, 2023-
Map releaseMar 15, 2023-
UpdateMar 22, 2023-
Current statusMar 22, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26989.png

Downloads & links

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Map

FileDownload / File: emd_26989.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationdeepEMhancer sharpened
Voxel sizeX=Y=Z: 0.8521 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.0010950407 - 5.1273956
Average (Standard dev.)0.0020150195 (±0.03476422)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions352352352
Spacing352352352
CellA=B=C: 299.9392 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26989_msk_1.map
Projections & Slices
AxesZYX

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Mask #2

Fileemd_26989_msk_2.map
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Additional map: unsharpened map

Fileemd_26989_additional_1.map
Annotationunsharpened map
Projections & Slices
AxesZYX

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Additional map: blurred map with 200 A2 B-factor applied, for Phenix refinement

Fileemd_26989_additional_2.map
Annotationblurred map with 200 A2 B-factor applied, for Phenix refinement
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AxesZYX

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Half map: half map B

Fileemd_26989_half_map_1.map
Annotationhalf map B
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Histogram

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Half map: half map A

Fileemd_26989_half_map_2.map
Annotationhalf map A
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Sample components

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Entire : haXWnt8-mFzd8CRD-hLRP6E1E2

EntireName: haXWnt8-mFzd8CRD-hLRP6E1E2
Components
  • Complex: haXWnt8-mFzd8CRD-hLRP6E1E2
    • Complex: Frizzled-8
      • Protein or peptide: Frizzled-8
    • Complex: Protein Wnt-8
      • Protein or peptide: Protein Wnt-8
    • Complex: Low-density lipoprotein receptor-related protein 6
      • Protein or peptide: Low-density lipoprotein receptor-related protein 6
  • Ligand: PALMITOLEIC ACID

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Supramolecule #1: haXWnt8-mFzd8CRD-hLRP6E1E2

SupramoleculeName: haXWnt8-mFzd8CRD-hLRP6E1E2 / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#3
Details: high-affinity XWnt8 variant in complex with mFzd8 CRD and hLRP6 E1E2
Molecular weightTheoretical: 130 KDa

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Supramolecule #2: Frizzled-8

SupramoleculeName: Frizzled-8 / type: complex / ID: 2 / Chimera: Yes / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #3: Protein Wnt-8

SupramoleculeName: Protein Wnt-8 / type: complex / ID: 3 / Chimera: Yes / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Xenopus laevis (African clawed frog)

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Supramolecule #4: Low-density lipoprotein receptor-related protein 6

SupramoleculeName: Low-density lipoprotein receptor-related protein 6 / type: complex / ID: 4 / Chimera: Yes / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Frizzled-8

MacromoleculeName: Frizzled-8 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 15.061343 KDa
Recombinant expressionOrganism: Drosophila melanogaster (fruit fly)
SequenceString:
ASAKELACQE ITVPLCKGIG YNYTYMPNQF NHDTQDEAGL EVHQFWPLVE IQCSPDLKFF LCSMYTPICL EDYKKPLPPC RSVCERAKA GCAPLMRQYG FAWPDRMRCD RLPEQGNPDT LCMDAAALEV LFQ

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Macromolecule #2: Protein Wnt-8

MacromoleculeName: Protein Wnt-8 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 36.67659 KDa
Recombinant expressionOrganism: Drosophila melanogaster (fruit fly)
SequenceString: GSAWSVNNFL MTGPKAYLTY SASVAVGAQN GIEECKYQFA WERWNCPEST LQLATHNGLR SATRETSFVH AISSAGVMYT LTRNCSMGD FDNCGCDDSR NGRIGGRGWV WGGCSDNAEF GERISKLFVD GLETGQDARA LMNLHNNEAG RLAVKETMKR T CKCHGISG ...String:
GSAWSVNNFL MTGPKAYLTY SASVAVGAQN GIEECKYQFA WERWNCPEST LQLATHNGLR SATRETSFVH AISSAGVMYT LTRNCSMGD FDNCGCDDSR NGRIGGRGWV WGGCSDNAEF GERISKLFVD GLETGQDARA LMNLHNNEAG RLAVKETMKR T CKCHGISG SCSIQTCWLQ LAEFRDIGNH LKIKHDQALK LEMDKRKMPS TNSVNSRRAI ADAFSSVAGS ELIFLEDSPD YC LKNISLG LQGTEGRECL QSGKNLSQWE RRSCKRLCTD CGLRVEEKKT EIISSCNCKF HWCCTVKCEQ CKQVVIKHFC ARH HHHHHH H

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Macromolecule #3: Low-density lipoprotein receptor-related protein 6

MacromoleculeName: Low-density lipoprotein receptor-related protein 6 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 73.384648 KDa
Recombinant expressionOrganism: Drosophila melanogaster (fruit fly)
SequenceString: GSAPLLLYAN RRDLRLVDAT NGKENATIVV GGLEDAAAVD FVFSHGLIYW SDVSEEAIKR TEFNKTESVQ NVVVSGLLSP DGLACDWLG EKLYWTDSET NRIEVSNLDG SLRKVLFWQE LDQPRAIALD PSSGFMYWTD WGEVPKIERA GMDGSSRFII I NSEIYWPN ...String:
GSAPLLLYAN RRDLRLVDAT NGKENATIVV GGLEDAAAVD FVFSHGLIYW SDVSEEAIKR TEFNKTESVQ NVVVSGLLSP DGLACDWLG EKLYWTDSET NRIEVSNLDG SLRKVLFWQE LDQPRAIALD PSSGFMYWTD WGEVPKIERA GMDGSSRFII I NSEIYWPN GLTLDYEEQK LYWADAKLNF IHKSNLDGTN RQAVVKGSLP HPFALTLFED ILYWTDWSTH SILACNKYTG EG LREIHSD IFSPMDIHAF SQQRQPNATN PCGIDNGGCS HLCLMSPVKP FYQCACPTGV KLLENGKTCK DGATELLLLA RRT DLRRIS LDTPDFTDIV LQLEDIRHAI AIDYDPVEGY IYWTDDEVRA IRRSFIDGSG SQFVVTAQIA HPDGIAVDWV ARNL YWTDT GTDRIEVTRL NGTMRKILIS EDLEEPRAIV LDPMVGYMYW TDWGEIPKIE RAALDGSDRV VLVNTSLGWP NGLAL DYDE GKIYWGDAKT DKIEVMNTDG TGRRVLVEDK IPHIFGFTLL GDYVYWTDWQ RRSIERVHKR SAEREVIIDQ LPDLMG LKA TNVHRVIGSN PCAEENGGCS HLCLYRPQGL RCACPIGFEL ISDMKTCIVS RGLEVLFQGP GAAGLNDIFE AQKIEWH EH HHHHHHH

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Macromolecule #4: PALMITOLEIC ACID

MacromoleculeName: PALMITOLEIC ACID / type: ligand / ID: 4 / Number of copies: 1 / Formula: PAM
Molecular weightTheoretical: 254.408 Da
Chemical component information

ChemComp-PAM:
PALMITOLEIC ACID / Palmitoleic acid

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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

Concentration0.5 mg/mL
BufferpH: 8
Component:
ConcentrationName
20.0 mMHepes-sodium salt
150.0 mMsodium chloride
2.0 mMcalcium chloride
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 3 s blotting before plunging.
DetailshaXWnt8-mFzd8CRD-hLRP6E1E2

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 58680 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 10077 / Average electron dose: 55.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3840364 / Details: Particles include duplicates and junk.
Startup modelType of model: OTHER
Details: The initial model was created by cryoSPARC ab initio reconstruction.
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3.3)
Final 3D classificationNumber classes: 50 / Software - Name: cryoSPARC (ver. 3.3)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3.3)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3)
Details: Model resolution is substantially lower and estimated to be ~10 angstrom based on the map-model FSC.
Number images used: 82235
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8ctg:
Extracellular architecture of an engineered canonical Wnt signaling ternary complex

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