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- EMDB-53969: FZD7 in complex with negative allosteric modulator C407 -

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Basic information

Entry
Database: EMDB / ID: EMD-53969
TitleFZD7 in complex with negative allosteric modulator C407
Map data
Sample
  • Complex: FZD7 in complex with the negative allosteric modulator C407
    • Protein or peptide: Frizzled-7
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: ethyl 3-[2-[(2-fluorophenyl)amino]-2-oxidanylidene-ethyl]-5-methyl-4-oxidanylidene-thieno[2,3-d]pyrimidine-6-carboxylate
KeywordsG protein-coupled receptor / Frizzled / negative allosteric modulator / small molecule / MEMBRANE PROTEIN
Function / homology
Function and homology information


negative regulation of ectodermal cell fate specification / negative regulation of cardiac muscle cell differentiation / somatic stem cell division / skeletal muscle satellite cell maintenance involved in skeletal muscle regeneration / non-canonical Wnt signaling pathway / Wnt receptor activity / mesenchymal to epithelial transition / positive regulation of epithelial cell proliferation involved in wound healing / Wnt-protein binding / WNT5:FZD7-mediated leishmania damping ...negative regulation of ectodermal cell fate specification / negative regulation of cardiac muscle cell differentiation / somatic stem cell division / skeletal muscle satellite cell maintenance involved in skeletal muscle regeneration / non-canonical Wnt signaling pathway / Wnt receptor activity / mesenchymal to epithelial transition / positive regulation of epithelial cell proliferation involved in wound healing / Wnt-protein binding / WNT5:FZD7-mediated leishmania damping / frizzled binding / PCP/CE pathway / Class B/2 (Secretin family receptors) / regulation of canonical Wnt signaling pathway / Wnt signaling pathway, planar cell polarity pathway / stem cell population maintenance / positive regulation of phosphorylation / negative regulation of cell-substrate adhesion / canonical Wnt signaling pathway / cellular response to retinoic acid / phosphatidylinositol-4,5-bisphosphate binding / substrate adhesion-dependent cell spreading / PDZ domain binding / Asymmetric localization of PCP proteins / positive regulation of JNK cascade / G protein-coupled receptor activity / recycling endosome membrane / neuron differentiation / T cell differentiation in thymus / positive regulation of MAPK cascade / intracellular membrane-bounded organelle / regulation of DNA-templated transcription / positive regulation of DNA-templated transcription / membrane / plasma membrane
Similarity search - Function
Frizzled-7, cysteine-rich Wnt-binding domain / 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. ...Frizzled-7, cysteine-rich Wnt-binding domain / 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. / GPCR, family 2-like / G-protein coupled receptors family 2 profile 2.
Similarity search - Domain/homology
Biological speciesSpodoptera frugiperda (fall armyworm) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsScharf MM / Graetz L / Kinsolving J / Voss J / Carrasco-Busturia D / Forsberg B / Kolb P / Schulte G
Funding support Sweden, Denmark, Germany, European Union, 14 items
OrganizationGrant numberCountry
Swedish Research Council2017-04676 Sweden
Swedish Research Council2019-01190 Sweden
Swedish Research Council2024-02515 Sweden
CancerfondenCAN2017/561 Sweden
Cancerfonden20 1102 PjF Sweden
Cancerfonden23 2825 Pj Sweden
Novo Nordisk FoundationNNF21OC0070008 Denmark
Novo Nordisk FoundationNNF22OC0078104 Denmark
German Research Foundation (DFG)504098926 Germany
German Research Foundation (DFG)470002134 Germany
German Research Foundation (DFG)520506488 Germany
Knut and Alice Wallenberg FoundationKAW 2020.0239 Sweden
European Union (EU)101199012European Union
Swedish Research Council2022-06725 Sweden
CitationJournal: Nat Commun / Year: 2025
Title: In silico docking yields small molecule negative allosteric modulators targeting the core of Frizzled 7.
Authors: Magdalena M Scharf / Julia Kinsolving / Lukas Grätz / Jan Hendrik Voss / David Carrasco-Busturia / Björn Forsberg / Peter Kolb / Gunnar Schulte /
Abstract: Targeting the Frizzled family (FZD) of WNT receptors pharmacologically has, despite substantial therapeutic potential, proven difficult. Given an almost complete lack of validated, effective small ...Targeting the Frizzled family (FZD) of WNT receptors pharmacologically has, despite substantial therapeutic potential, proven difficult. Given an almost complete lack of validated, effective small molecules targeting FZDs, no putative ligand binding site has so far been identified. In order to target FZD, a potential target for the treatment of intestinal tumors, we combine an approach of adapted docking setups and large molecular library docking screens, identifying compound C407. Applying pharmacological assays, genetically-encoded biosensors, site-directed mutagenesis, cryo-electron microscopy and molecular dynamics simulations, the compound binding site in the core of the seven transmembrane bundle is validated and C407 is confirmed as a negative allosteric modulator of WNT-induced and FZD-mediated WNT/β-catenin signaling. In summary, we provide here the proof-of-principle that targeting FZDs with small molecule compounds is possible and effective. Future hit optimization and functional validation in disease-relevant in vitro and in vivo models will pave the way towards clinical exploration.
History
DepositionJun 9, 2025-
Header (metadata) releaseDec 24, 2025-
Map releaseDec 24, 2025-
UpdateDec 24, 2025-
Current statusDec 24, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_53969.png

Downloads & links

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Map

FileDownload / File: emd_53969.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 250 pix.
= 261.25 Å		1.05 Å/pix.
x 250 pix.
= 261.25 Å		1.05 Å/pix.
x 250 pix.
= 261.25 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.045 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.433
Minimum - Maximum-2.9879773 - 3.2561362
Average (Standard dev.)-0.0011400965 (±0.080918096)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 261.25 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_53969_msk_1.map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_53969_half_map_1.map
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Histogram

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Half map: #1

Fileemd_53969_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : FZD7 in complex with the negative allosteric modulator C407

EntireName: FZD7 in complex with the negative allosteric modulator C407
Components
  • Complex: FZD7 in complex with the negative allosteric modulator C407
    • Protein or peptide: Frizzled-7
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: ethyl 3-[2-[(2-fluorophenyl)amino]-2-oxidanylidene-ethyl]-5-methyl-4-oxidanylidene-thieno[2,3-d]pyrimidine-6-carboxylate

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Supramolecule #1: FZD7 in complex with the negative allosteric modulator C407

SupramoleculeName: FZD7 in complex with the negative allosteric modulator C407
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Spodoptera frugiperda (fall armyworm)

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Macromolecule #1: Frizzled-7

MacromoleculeName: Frizzled-7 / type: protein_or_peptide / ID: 1
Details: Expression tag position 1-24, expression tag 578-605
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 67.460859 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MKTIIALSYI FCLVFADYKD DDDQPYHGEK GISVPDHGFC QPISIPLCTD IAYNQTILPN LLGHTNQEDA GLEVHQFYPL VKVQCSPEL RFFLCSMYAP VCTVLDQAIP PCRSLCERAR QGCEALMNKF GFQWPERLRC ENFPVHGAGE ICVGQNTSDG S GGPGGGPT ...String:
MKTIIALSYI FCLVFADYKD DDDQPYHGEK GISVPDHGFC QPISIPLCTD IAYNQTILPN LLGHTNQEDA GLEVHQFYPL VKVQCSPEL RFFLCSMYAP VCTVLDQAIP PCRSLCERAR QGCEALMNKF GFQWPERLRC ENFPVHGAGE ICVGQNTSDG S GGPGGGPT AYPTAPYLPD LPFTALPPGA SDGRGRPAFP FSCPRQLKVP PYLGYRFLGE RDCGAPCEPG RANGLMYFKE EE RRFARLW VGVWSVLCCA STLFTVLTYL VDMRRFSYPE RPIIFLSGCY FMVAVAHVAG FLLEDRAVCV ERFSDDGYRT VAQ GTKKEG CTILFMVLYF FGMASSIWWV ILSLTWFLAA GMKWGHEAIE ANSQYFHLAA WAVPAVKTIT ILAMGQVDGD LLSG VCYVG LSSVDALRGF VLAPLFVYLF IGTSFLLAGF VSLFRIRTIM KHDGTKTEKL EKLMVRIGVF SVLYTVPATI VLACY FYEQ AFREHWERTW LLQTCKSYAV PCPPGHFPPM SPDFTVFMIK YLMTMIVGIT TGFWIWSGKT LQSWRRFYHR LSHSSK GET AVSRLEVLFQ GPWSHPQFEK GGGSGGGSGG GSWSHPQFEK

UniProtKB: Frizzled-7

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Macromolecule #2: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 2 / Number of copies: 4 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #3: ethyl 3-[2-[(2-fluorophenyl)amino]-2-oxidanylidene-ethyl]-5-methy...

MacromoleculeName: ethyl 3-[2-[(2-fluorophenyl)amino]-2-oxidanylidene-ethyl]-5-methyl-4-oxidanylidene-thieno[2,3-d]pyrimidine-6-carboxylate
type: ligand / ID: 3 / Number of copies: 1 / Formula: A1JGQ
Molecular weightTheoretical: 389.401 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

Concentration2.88 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
10.0 mMTRISTRIS
100.0 mMNaClsodium chloride
0.002 %LMNGlauryl maltose neopentyl glycol
0.0002 %CHScholesterol hemisuccinate
0.0002 %GDNglyco-diosgenin
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
DetailsPurified protein sample was supplemented with a 10:1 molar ratio of C407 (prepared in DMSO) to FZD7 prior to grid freezing.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 19899 / Average exposure time: 1.4 sec. / Average electron dose: 80.1 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 165000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionDetails: Blob picker
CTF correctionSoftware - Name: cryoSPARC (ver. v4.7.0) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

9epo

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4.7.0) / Number images used: 191045
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.7.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.7.0)
Final 3D classificationSoftware - Name: cryoSPARC (ver. v4.7.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

9epo


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-9rhg:
FZD7 in complex with negative allosteric modulator C407

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