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- EMDB-21148: Cryo-EM structure of the Dvl2 DIX filament -

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Basic information

Entry
Database: EMDB / ID: EMD-21148
TitleCryo-EM structure of the Dvl2 DIX filament
Map dataDvl2 DIX filament
Sample
  • Complex: Segment polarity protein dishevelled homolog DVL-2
    • Protein or peptide: Segment polarity protein dishevelled homolog DVL-2
KeywordsDishevelled / DIX domain / Wnt signaling / SIGNALING PROTEIN
Function / homology
Function and homology information


: / WNT mediated activation of DVL / convergent extension involved in organogenesis / PCP/CE pathway / convergent extension involved in neural plate elongation / Signaling by Hippo / Disassembly of the destruction complex and recruitment of AXIN to the membrane / segmentation / planar cell polarity pathway involved in neural tube closure / Asymmetric localization of PCP proteins ...: / WNT mediated activation of DVL / convergent extension involved in organogenesis / PCP/CE pathway / convergent extension involved in neural plate elongation / Signaling by Hippo / Disassembly of the destruction complex and recruitment of AXIN to the membrane / segmentation / planar cell polarity pathway involved in neural tube closure / Asymmetric localization of PCP proteins / cochlea morphogenesis / WNT5A-dependent internalization of FZD4 / Degradation of DVL / segment specification / non-canonical Wnt signaling pathway / positive regulation of neuron projection arborization / RHO GTPases Activate Formins / 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 / canonical Wnt signaling pathway / lateral plasma membrane / heart morphogenesis / positive regulation of JUN kinase activity / neural tube closure / positive regulation of JNK cascade / protein localization / small GTPase binding / Wnt signaling pathway / positive regulation of GTPase activity / : / protein-macromolecule adaptor activity / apical part of cell / cell cortex / heart development / cytoplasmic vesicle / nuclear body / cytoskeleton / intracellular signal transduction / positive regulation of protein phosphorylation / protein domain specific binding / regulation of DNA-templated transcription / protein kinase binding / positive regulation of transcription by RNA polymerase II / nucleoplasm / identical protein binding / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
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 ...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 / 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
Similarity search - Domain/homology
Segment polarity protein dishevelled homolog DVL-2
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodhelical reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsEnos M / Kan W
Funding support United States, 1 items
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, 2019-
Header (metadata) releaseJan 29, 2020-
Map releaseApr 29, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6vcc
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21148.png

Downloads & links

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Map

FileDownload / File: emd_21148.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDvl2 DIX filament
Voxel sizeX=Y=Z: 1 Å
Density
Contour LevelBy AUTHOR: 0.0296 / Movie #1: 0.035
Minimum - Maximum-0.16656485 - 0.23581737
Average (Standard dev.)-0.00003109892 (±0.010861889)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions150150150
Spacing150150150
CellA=B=C: 150.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z150.000150.000150.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ350350350
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS150150150
D min/max/mean-0.1670.236-0.000

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Supplemental data

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Mask #1

Fileemd_21148_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_21148_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_21148_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Segment polarity protein dishevelled homolog DVL-2

EntireName: Segment polarity protein dishevelled homolog DVL-2
Components
  • Complex: Segment polarity protein dishevelled homolog DVL-2
    • Protein or peptide: Segment polarity protein dishevelled homolog DVL-2

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Supramolecule #1: Segment polarity protein dishevelled homolog DVL-2

SupramoleculeName: Segment polarity protein dishevelled homolog DVL-2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Protein spontaneously formed a double-stranded, antiparallel helical filament upon removal of the MBP tag with TEV protease.
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Segment polarity protein dishevelled homolog DVL-2

MacromoleculeName: Segment polarity protein dishevelled homolog DVL-2 / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 9.333547 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
GSGETKVIYH LDEEETPYLV KIPVPAERIT LGDFKSVLQR PAGAKYFFKS MDQDFGVVKE EISDDNARLP CFNGRVVSWL VSS

UniProtKB: Segment polarity protein dishevelled homolog DVL-2

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

Concentration0.12 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chloridesodium chloride
4.0 mMC4H10O2S2dithiothreitol
1.0 mMC10H16N2O8EDTAEthylenediaminetetraacetic acid

Details: Dithiothreitol was added fresh the day of use.
GridMaterial: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.04 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 295 K / Instrument: LEICA EM GP
Details: The grid was blotted for 2.0 s using Whatman #1 filter paper (GE Healthcare)..
DetailsSample was taken from a single fraction of a gel filtration run performed immediately before grid preparation.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.0 µm / Calibrated defocus min: 0.5 µm / Calibrated magnification: 29000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 4-50 / Number real images: 540 / Average exposure time: 10.0 sec. / Average electron dose: 98.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 437872
Startup modelType of model: INSILICO MODEL
In silico model: A starting model was generated using the stochastic gradient descent option from the "Initial model" jobtype in RELION 3.0.8.
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 3.0.8)
Final reconstructionNumber classes used: 2
Applied symmetry - Helical parameters - Δz: 13.5 Å
Applied symmetry - Helical parameters - Δ&Phi: 48.0 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.8) / Number images used: 110105
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6iw3


Chain - Chain ID: A / Chain - Residue range: 12-92 / Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsThe 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.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

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

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