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- PDB-8q7o: Crystal structure of the FZD3 cysteine-rich domain in complex wit... -

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Basic information

Entry
Database: PDB / ID: 8q7o
TitleCrystal structure of the FZD3 cysteine-rich domain in complex with a nanobody (14478)
Components
  • Frizzled-3
  • Nanobody 14478
KeywordsSIGNALING PROTEIN / Frizzled / WNT / GPCR / Nanobody / Complex
Function / homology
Function and homology information


dopaminergic neuron axon guidance / serotonergic neuron axon guidance / cell proliferation in midbrain / establishment of planar polarity / negative regulation of mitotic cell cycle, embryonic / Wnt receptor activity / midbrain morphogenesis / motor neuron migration / non-canonical Wnt signaling pathway / sympathetic ganglion development ...dopaminergic neuron axon guidance / serotonergic neuron axon guidance / cell proliferation in midbrain / establishment of planar polarity / negative regulation of mitotic cell cycle, embryonic / Wnt receptor activity / midbrain morphogenesis / motor neuron migration / non-canonical Wnt signaling pathway / sympathetic ganglion development / filopodium tip / Wnt-protein binding / post-anal tail morphogenesis / negative regulation of execution phase of apoptosis / commissural neuron axon guidance / PCP/CE pathway / Class B/2 (Secretin family receptors) / Wnt signaling pathway, planar cell polarity pathway / inner ear morphogenesis / positive regulation of neuroblast proliferation / presynaptic active zone / hair follicle development / lateral plasma membrane / canonical Wnt signaling pathway / response to electrical stimulus / neural tube closure / Asymmetric localization of PCP proteins / PDZ domain binding / G protein-coupled receptor activity / neuron differentiation / Ca2+ pathway / response to xenobiotic stimulus / apical plasma membrane / axon / neuronal cell body / dendrite / cell surface / plasma membrane / cytoplasm
Similarity search - Function
Frizzled 3, cysteine-rich 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 3, cysteine-rich 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
CITRIC ACID / Frizzled-3
Similarity search - Component
Biological speciesLama glama (llama)
Homo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.76 Å
AuthorsHillier, J.S. / Zhao, Y. / Malinauskas, T. / Jones, E.Y.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Cancer Research UK United Kingdom
CitationJournal: Nat Commun / Year: 2024
Title: Structural insights into Frizzled3 through nanobody modulators.
Authors: James Hillier / Yuguang Zhao / Loic Carrique / Tomas Malinauskas / Reinis R Ruza / Tao-Hsin Chang / Gangshun Yi / Helen M E Duyvesteyn / Jing Yu / Weixian Lu / Els Pardon / Jan Steyaert / ...Authors: James Hillier / Yuguang Zhao / Loic Carrique / Tomas Malinauskas / Reinis R Ruza / Tao-Hsin Chang / Gangshun Yi / Helen M E Duyvesteyn / Jing Yu / Weixian Lu / Els Pardon / Jan Steyaert / Yanan Zhu / Tao Ni / E Yvonne Jones /
Abstract: The Wnt receptor Frizzled3 (FZD3) is important for brain axonal development and cancer progression. We report structures of FZD3 in complex with extracellular and intracellular binding nanobodies (Nb) ...The Wnt receptor Frizzled3 (FZD3) is important for brain axonal development and cancer progression. We report structures of FZD3 in complex with extracellular and intracellular binding nanobodies (Nb). The crystal structure of Nb8 in complex with the FZD3 cysteine-rich domain (CRD) reveals that the nanobody binds at the base of the lipid-binding groove and can compete with Wnt5a. Nb8 fused with the Dickkopf-1 C-terminal domain behaves as a FZD3-specific Wnt surrogate, activating β-catenin signalling. The cryo-EM structure of FZD3 in complex with Nb9 reveals partially resolved density for the CRD, which exhibits positional flexibility, and a transmembrane conformation that resembles active GPCRs. Nb9 binds to the cytoplasmic region of FZD3 at the putative Dishevelled (DVL) or G protein-binding site, competes with DVL binding, and inhibits GαS coupling. In combination, our FZD3 structures with nanobody modulators map extracellular and intracellular interaction surfaces of functional, and potentially therapeutic, relevance.
History
DepositionAug 16, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 28, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 4, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Nanobody 14478
B: Nanobody 14478
C: Frizzled-3
D: Frizzled-3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)56,79015
Polymers55,4354
Non-polymers1,35511
Water3,729207
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6780 Å2
ΔGint-68 kcal/mol
Surface area25960 Å2
MethodPISA
Unit cell
Length a, b, c (Å)53.659, 147.475, 229.727
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221
Components on special symmetry positions
IDModelComponents
11B-350-

HOH

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Components

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Antibody / Protein / Sugars , 3 types, 6 molecules ABCD

#1: Antibody Nanobody 14478


Mass: 13264.633 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Cell line (production host): HEK293S GnTI- / Production host: Homo sapiens (human)
#2: Protein Frizzled-3 / Fz-3 / hFz3


Mass: 14452.656 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FZD3 / Cell line (production host): HEK293S GnTI- / Production host: Homo sapiens (human) / References: UniProt: Q9NPG1
#6: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 5 types, 216 molecules

#3: Chemical ChemComp-DMS / DIMETHYL SULFOXIDE


Mass: 78.133 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H6OS / Comment: DMSO, precipitant*YM
#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H8O3
#5: Chemical ChemComp-CIT / CITRIC ACID


Mass: 192.124 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H8O7
#7: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Formula: SO4
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 207 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop
Details: 100 mM citric acid, pH 3.6; 690 mM ammonium sulphate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.9762 Å
DetectorType: DECTRIS EIGER2 XE 16M / Detector: PIXEL / Date: Apr 28, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9762 Å / Relative weight: 1
ReflectionResolution: 1.759→46.17 Å / Num. obs: 90730 / % possible obs: 99.51 % / Redundancy: 13.8 % / CC1/2: 0.999 / Net I/σ(I): 15.4
Reflection shellResolution: 1.759→1.822 Å / Num. unique obs: 8825 / CC1/2: 0.797

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Processing

SoftwareName: PHENIX / Version: (1.19.2_4158: ???) / Classification: refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.76→46.17 Å / SU ML: 0.21 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 29.09 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2222 4520 5 %
Rwork0.2172 --
obs0.2174 90453 99.52 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.76→46.17 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3792 0 82 207 4081
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0073957
X-RAY DIFFRACTIONf_angle_d0.8875367
X-RAY DIFFRACTIONf_dihedral_angle_d14.3171448
X-RAY DIFFRACTIONf_chiral_restr0.057571
X-RAY DIFFRACTIONf_plane_restr0.008697
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.76-1.780.42141320.38332694X-RAY DIFFRACTION94
1.78-1.80.3831560.36882802X-RAY DIFFRACTION99
1.8-1.820.33711680.35132804X-RAY DIFFRACTION99
1.82-1.840.34641500.32682838X-RAY DIFFRACTION100
1.84-1.870.33141600.3162837X-RAY DIFFRACTION100
1.87-1.890.36651400.31042786X-RAY DIFFRACTION99
1.89-1.920.31421740.3092859X-RAY DIFFRACTION99
1.92-1.950.31551530.28912791X-RAY DIFFRACTION99
1.95-1.980.25681390.27332874X-RAY DIFFRACTION100
1.98-2.010.24061410.26162837X-RAY DIFFRACTION99
2.01-2.050.27181370.23482858X-RAY DIFFRACTION100
2.05-2.090.24361560.22532822X-RAY DIFFRACTION100
2.09-2.130.22681500.2262874X-RAY DIFFRACTION100
2.13-2.170.25451270.22262854X-RAY DIFFRACTION100
2.17-2.220.23011650.21882822X-RAY DIFFRACTION100
2.22-2.270.2291600.22372859X-RAY DIFFRACTION100
2.27-2.320.23351520.22982862X-RAY DIFFRACTION100
2.32-2.390.26331500.22432855X-RAY DIFFRACTION100
2.39-2.460.24711370.2292900X-RAY DIFFRACTION100
2.46-2.540.25431370.22712885X-RAY DIFFRACTION100
2.54-2.630.25961360.23062855X-RAY DIFFRACTION100
2.63-2.730.27321350.22542921X-RAY DIFFRACTION100
2.73-2.860.24431530.2312851X-RAY DIFFRACTION100
2.86-3.010.25131570.24192902X-RAY DIFFRACTION100
3.01-3.20.23961500.23732894X-RAY DIFFRACTION100
3.2-3.440.23171640.22212882X-RAY DIFFRACTION100
3.44-3.790.22691520.21472926X-RAY DIFFRACTION100
3.79-4.340.18861590.17332940X-RAY DIFFRACTION100
4.34-5.460.15541540.16082967X-RAY DIFFRACTION100
5.46-46.170.16591760.20433082X-RAY DIFFRACTION100
Refinement TLS params.Method: refined / Origin x: 13.9767 Å / Origin y: 30.8473 Å / Origin z: 25.3798 Å
111213212223313233
T0.3149 Å20.0039 Å2-0.0046 Å2-0.3231 Å20.0199 Å2--0.3118 Å2
L0.0718 °2-0.0245 °2-0.1507 °2-0.2071 °20.001 °2--0.3597 °2
S-0.0644 Å °-0.053 Å °-0.0338 Å °0.0972 Å °0.0124 Å °-0.0492 Å °0.0649 Å °0.0629 Å °0.0482 Å °
Refinement TLS groupSelection details: all

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