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- PDB-6vcc: Cryo-EM structure of the Dvl2 DIX filament -

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Basic information

Entry
Database: PDB / ID: 6vcc
TitleCryo-EM structure of the Dvl2 DIX filament
ComponentsSegment polarity protein dishevelled homolog DVL-2
KeywordsSIGNALING PROTEIN / Dishevelled / DIX domain / Wnt signaling
Function / homology
Function and homology information


WNT mediated activation of DVL / convergent extension involved in organogenesis / PCP/CE pathway / Signaling by Hippo / convergent extension involved in neural plate elongation / segmentation / Disassembly of the destruction complex and recruitment of AXIN to the membrane / : / cochlea morphogenesis / Asymmetric localization of PCP proteins ...WNT mediated activation of DVL / convergent extension involved in organogenesis / PCP/CE pathway / Signaling by Hippo / convergent extension involved in neural plate elongation / segmentation / Disassembly of the destruction complex and recruitment of AXIN to the membrane / : / cochlea morphogenesis / Asymmetric localization of PCP proteins / WNT5A-dependent internalization of FZD4 / Degradation of DVL / segment specification / RHO GTPases Activate Formins / positive regulation of neuron projection arborization / Cargo recognition for clathrin-mediated endocytosis / Clathrin-mediated endocytosis / frizzled binding / aggresome / Wnt signaling pathway, planar cell polarity pathway / clathrin-coated vesicle / heart looping / outflow tract morphogenesis / lateral plasma membrane / canonical Wnt signaling pathway / heart morphogenesis / positive regulation of JUN kinase activity / positive regulation of GTPase activity / neural tube closure / positive regulation of JNK cascade / Wnt signaling pathway / small GTPase binding / protein localization / apical part of cell / heart development / regulation of cell population proliferation / protein-macromolecule adaptor activity / cell cortex / cytoplasmic vesicle / cytoskeleton / nuclear body / intracellular signal transduction / protein domain specific binding / protein kinase binding / positive regulation of transcription by RNA polymerase II / identical protein binding / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Ribosomal Protein L25; Chain P - #130 / Dishevelled-2 / Dishevelled protein domain / Dishevelled family / Dishevelled C-terminal / Dishevelled specific domain / Segment polarity protein dishevelled (Dsh) C terminal / Dishevelled-related protein / DIX domain / DIX domain superfamily ...Ribosomal Protein L25; Chain P - #130 / Dishevelled-2 / Dishevelled protein domain / Dishevelled family / Dishevelled C-terminal / Dishevelled specific domain / Segment polarity protein dishevelled (Dsh) C terminal / Dishevelled-related protein / DIX domain / DIX domain superfamily / DIX domain / DIX domain profile. / Domain present in Dishevelled and axin / Ribosomal Protein L25; Chain P / Domain found in Dishevelled, Egl-10, and Pleckstrin (DEP) / DEP domain profile. / Domain found in Dishevelled, Egl-10, and Pleckstrin / DEP domain / PDZ domain / PDZ domain profile. / Domain present in PSD-95, Dlg, and ZO-1/2. / PDZ domain / PDZ superfamily / Ubiquitin-like domain superfamily / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / Beta Barrel / Mainly Beta
Similarity search - Domain/homology
Segment polarity protein dishevelled homolog DVL-2
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsEnos, M. / Kan, W. / Muennich, S. / Chen, D.H. / Skiniotis, G. / Weis, W.I.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM131747 United States
CitationJournal: Elife / Year: 2020
Title: Limited dishevelled/Axin oligomerization determines efficiency of Wnt/β-catenin signal transduction.
Authors: Wei Kan / Michael D Enos / Elgin Korkmazhan / Stefan Muennich / Dong-Hua Chen / Melissa V Gammons / Mansi Vasishtha / Mariann Bienz / Alexander R Dunn / Georgios Skiniotis / William I Weis /
Abstract: In Wnt/β-catenin signaling, the transcriptional coactivator β-catenin is regulated by its phosphorylation in a complex that includes the scaffold protein Axin and associated kinases. Wnt binding to ...In Wnt/β-catenin signaling, the transcriptional coactivator β-catenin is regulated by its phosphorylation in a complex that includes the scaffold protein Axin and associated kinases. Wnt binding to its coreceptors activates the cytosolic effector Dishevelled (Dvl), leading to the recruitment of Axin and the inhibition of β-catenin phosphorylation. This process requires interaction of homologous DIX domains present in Dvl and Axin, but is mechanistically undefined. We show that Dvl DIX forms antiparallel, double-stranded oligomers in vitro, and that Dvl in cells forms oligomers typically <10 molecules at endogenous expression levels. Axin DIX (DAX) forms small single-stranded oligomers, but its self-association is stronger than that of DIX. DAX caps the ends of DIX oligomers, such that a DIX oligomer has at most four DAX binding sites. The relative affinities and stoichiometry of the DIX-DAX interaction provide a mechanism for efficient inhibition of β-catenin phosphorylation upon Axin recruitment to the Wnt receptor complex.
History
DepositionDec 20, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 29, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: Segment polarity protein dishevelled homolog DVL-2
B: Segment polarity protein dishevelled homolog DVL-2
C: Segment polarity protein dishevelled homolog DVL-2
D: Segment polarity protein dishevelled homolog DVL-2
E: Segment polarity protein dishevelled homolog DVL-2
F: Segment polarity protein dishevelled homolog DVL-2
G: Segment polarity protein dishevelled homolog DVL-2
H: Segment polarity protein dishevelled homolog DVL-2
I: Segment polarity protein dishevelled homolog DVL-2
J: Segment polarity protein dishevelled homolog DVL-2
K: Segment polarity protein dishevelled homolog DVL-2
L: Segment polarity protein dishevelled homolog DVL-2


Theoretical massNumber of molelcules
Total (without water)112,00312
Polymers112,00312
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, microscopy, Cryo-electron microscopy., assay for oligomerization, Sedimentation as a function of concentration.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area13620 Å2
ΔGint-45 kcal/mol
Surface area48970 Å2
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 6 / Rise per n subunits: 13.5 Å / Rotation per n subunits: 40 °)

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Components

#1: Protein
Segment polarity protein dishevelled homolog DVL-2 / Dishevelled-2 / DSH homolog 2


Mass: 9333.547 Da / Num. of mol.: 12 / Fragment: DIX domain (UNP residues 12-92)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Dvl2 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): RIL / References: UniProt: Q60838

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Segment polarity protein dishevelled homolog DVL-2 / Type: COMPLEX
Details: Protein spontaneously formed a double-stranded, antiparallel helical filament upon removal of the MBP tag with TEV protease.
Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: RIL / Plasmid: pCDF-Duet
Buffer solutionpH: 8 / Details: Dithiothreitol was added fresh the day of use.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaCl1
34 mMdithiothreitolC4H10O2S21
41 mMEDTAC10H16N2O81
SpecimenConc.: 0.12 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was taken from a single fraction of a gel filtration run performed immediately before grid preparation.
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in.
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 295 K
Details: The grid was blotted for 2.0 s using Whatman #1 filter paper (GE Healthcare).

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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 FIELD / Calibrated magnification: 29000 X / Calibrated defocus min: 500 nm / Calibrated defocus max: 2000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 98 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 540
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 50 / Used frames/image: 4-50

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
3DigitalMicrographimage acquisition
5CTFFIND4.1CTF correction
8PHENIX1.17.1-3660model fitting
10RELION3.0.8initial Euler assignment
11RELION3.0.8final Euler assignment
12RELION3.0.8classification
13RELION3.0.83D reconstruction
14PHENIX1.17.1-3360model refinement
15Coot0.8.9.2 ELmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 48 ° / Axial rise/subunit: 13.5 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 437872
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 110105 / Algorithm: BACK PROJECTION / Num. of class averages: 2 / Symmetry type: HELICAL
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: The two mutations in the source (Y27W and C80S) were reverted to Trp and Ser, respectively, before fitting. Model refinement was carried out through alternating rounds of real-space ...Details: The two mutations in the source (Y27W and C80S) were reverted to Trp and Ser, respectively, before fitting. Model refinement was carried out through alternating rounds of real-space refinement in PHENIX and manual building in Coot.
Atomic model buildingPDB-ID: 6IW3

6iw3


Pdb chain-ID: A / Accession code: 6IW3 / Pdb chain residue range: 12-92 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037800
ELECTRON MICROSCOPYf_angle_d0.61910560
ELECTRON MICROSCOPYf_dihedral_angle_d6.7671020
ELECTRON MICROSCOPYf_chiral_restr0.0541152
ELECTRON MICROSCOPYf_plane_restr0.0061368

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