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- EMDB-22806: Human WLS in complex with WNT8A -

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Basic information

Entry
Database: EMDB / ID: EMD-22806
TitleHuman WLS in complex with WNT8A
Map dataDensity modified map
Sample
  • Complex: Complex of WLS/Evi and WNT8A in nanodisc
    • Protein or peptide: Protein Wnt-8a
    • Protein or peptide: Protein wntless homolog
  • Ligand: PALMITIC ACID
  • Ligand: 1-CIS-9-OCTADECANOYL-2-CIS-9-HEXADECANOYL PHOSPHATIDYL GLYCEROL
  • Ligand: CHOLESTEROL HEMISUCCINATE
Function / homology
Function and homology information


: / : / Wnt protein secretion / neural crest cell fate commitment / positive regulation of Wnt protein secretion / WNT ligand biogenesis and trafficking / secondary palate development / cementum mineralization / beta-catenin destruction complex disassembly / hindbrain development ...: / : / Wnt protein secretion / neural crest cell fate commitment / positive regulation of Wnt protein secretion / WNT ligand biogenesis and trafficking / secondary palate development / cementum mineralization / beta-catenin destruction complex disassembly / hindbrain development / Wnt-protein binding / exocrine pancreas development / frizzled binding / Class B/2 (Secretin family receptors) / Disassembly of the destruction complex and recruitment of AXIN to the membrane / anterior/posterior axis specification / midbrain development / mesoderm formation / positive regulation of Wnt signaling pathway / canonical Wnt signaling pathway / endomembrane system / cell fate commitment / response to retinoic acid / TCF dependent signaling in response to WNT / cytokine activity / intracellular protein transport / trans-Golgi network / neuron differentiation / Wnt signaling pathway / positive regulation of canonical Wnt signaling pathway / endocytic vesicle membrane / cytoplasmic vesicle / early endosome membrane / collagen-containing extracellular matrix / positive regulation of canonical NF-kappaB signal transduction / receptor ligand activity / early endosome / Golgi membrane / endoplasmic reticulum membrane / Golgi apparatus / endoplasmic reticulum / positive regulation of transcription by RNA polymerase II / extracellular space / extracellular exosome / extracellular region / identical protein binding / plasma membrane / cytosol
Similarity search - Function
Wnt-8 protein / Protein Wnt-8A/8C / Protein wntless / : / Wnt-binding factor required for Wnt secretion / Wnt protein, conserved site / Wnt-1 family signature. / Wnt / Wnt, C-terminal domain / wnt family / found in Wnt-1
Similarity search - Domain/homology
Protein wntless homolog / Protein Wnt-8a
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.19 Å
AuthorsNygaard R / Jia Y / Kim J / Ross D / Parisi G / Clarke OB / Virshup DM / Mancia F
Funding support United States, Singapore, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM132120 United States
National Research Foundation (NRF, Singapore)MOH-000155 Singapore
CitationJournal: Cell / Year: 2021
Title: Structural Basis of WLS/Evi-Mediated Wnt Transport and Secretion.
Authors: Rie Nygaard / Jia Yu / Jonathan Kim / Daniel R Ross / Giacomo Parisi / Oliver B Clarke / David M Virshup / Filippo Mancia /
Abstract: Wnts are evolutionarily conserved ligands that signal at short range to regulate morphogenesis, cell fate, and stem cell renewal. The first and essential steps in Wnt secretion are their O- ...Wnts are evolutionarily conserved ligands that signal at short range to regulate morphogenesis, cell fate, and stem cell renewal. The first and essential steps in Wnt secretion are their O-palmitoleation and subsequent loading onto the dedicated transporter Wntless/evenness interrupted (WLS/Evi). We report the 3.2 Å resolution cryogenic electron microscopy (cryo-EM) structure of palmitoleated human WNT8A in complex with WLS. This is accompanied by biochemical experiments to probe the physiological implications of the observed association. The WLS membrane domain has close structural homology to G protein-coupled receptors (GPCRs). A Wnt hairpin inserts into a conserved hydrophobic cavity in the GPCR-like domain, and the palmitoleate protrudes between two helices into the bilayer. A conformational switch of highly conserved residues on a separate Wnt hairpin might contribute to its transfer to receiving cells. This work provides molecular-level insights into a central mechanism in animal body plan development and stem cell biology.
History
DepositionOct 5, 2020-
Header (metadata) releaseJan 6, 2021-
Map releaseJan 6, 2021-
UpdateJan 20, 2021-
Current statusJan 20, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.463
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.463
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7kc4
  • Surface level: 0.463
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22806.png

Downloads & links

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Map

FileDownload / File: emd_22806.map.gz / Format: CCP4 / Size: 9.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDensity modified map
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.463 / Movie #1: 0.463
Minimum - Maximum-2.3511496 - 4.150369
Average (Standard dev.)-2.5291134e-12 (±0.22323276)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions122121175
Spacing121122175
CellA: 100.43 Å / B: 101.259995 Å / C: 145.25 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.830.830.83
M x/y/z121122175
origin x/y/z0.0000.0000.000
length x/y/z100.430101.260145.250
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS121122175
D min/max/mean-2.3514.150-0.000

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Supplemental data

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Mask #1

Fileemd_22806_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Mask #2

Fileemd_22806_msk_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Blurred map (B=100)

Fileemd_22806_additional_1.map
AnnotationBlurred map (B=100)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map

Fileemd_22806_half_map_1.map
AnnotationHalf map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map

Fileemd_22806_half_map_2.map
AnnotationHalf 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 : Complex of WLS/Evi and WNT8A in nanodisc

EntireName: Complex of WLS/Evi and WNT8A in nanodisc
Components
  • Complex: Complex of WLS/Evi and WNT8A in nanodisc
    • Protein or peptide: Protein Wnt-8a
    • Protein or peptide: Protein wntless homolog
  • Ligand: PALMITIC ACID
  • Ligand: 1-CIS-9-OCTADECANOYL-2-CIS-9-HEXADECANOYL PHOSPHATIDYL GLYCEROL
  • Ligand: CHOLESTEROL HEMISUCCINATE

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Supramolecule #1: Complex of WLS/Evi and WNT8A in nanodisc

SupramoleculeName: Complex of WLS/Evi and WNT8A in nanodisc / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 / Details: Nanodisc were formed using MSP1E3D1 and POPG lipid
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant strain: FreeStyle 293-F Cells
Molecular weightTheoretical: 105.6 KDa

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Macromolecule #1: Protein Wnt-8a

MacromoleculeName: Protein Wnt-8a / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 40.052359 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGNLFMLWAA LGICCAAFSA SAWSVNNFLI TGPKAYLTYT TSVALGAQSG IEECKFQFAW ERWNCPENAL QLSTHNRLRS ATRETSFIH AISSAGVMYI ITKNCSMGDF ENCGCDGSNN GKTGGHGWIW GGCSDNVEFG ERISKLFVDS LEKGKDARAL M NLHNNRAG ...String:
MGNLFMLWAA LGICCAAFSA SAWSVNNFLI TGPKAYLTYT TSVALGAQSG IEECKFQFAW ERWNCPENAL QLSTHNRLRS ATRETSFIH AISSAGVMYI ITKNCSMGDF ENCGCDGSNN GKTGGHGWIW GGCSDNVEFG ERISKLFVDS LEKGKDARAL M NLHNNRAG RLAVRATMKR TCKCHGISGS CSIQTCWLQL AEFREMGDYL KAKYDQALKI EMDKRQLRAG NSAEGHWVPA EA FLPSAEA ELIFLEESPD YCTCNSSLGI YGTEGRECLQ NSHNTSRWER RSCGRLCTEC GLQVEERKTE VISSCNCKFQ WCC TVKCDQ CRHVVSKYYC ARSPGSAQSL GKGSAGGWSH PQFEK

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Macromolecule #2: Protein wntless homolog

MacromoleculeName: Protein wntless homolog / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 65.64943 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAGAIIENMS TKKLCIVGGI LLVFQIIAFL VGGLIAPGPT TAVSYMSVKC VDARKNHHKT KWFVPWGPNH CDKIRDIEEA IPREIEAND IVFSVHIPLP HMEMSPWFQF MLFILQLDIA FKLNNQIREN AEVSMDVSLA YRDDAFAEWT EMAHERVPRK L KCTFTSPK ...String:
MAGAIIENMS TKKLCIVGGI LLVFQIIAFL VGGLIAPGPT TAVSYMSVKC VDARKNHHKT KWFVPWGPNH CDKIRDIEEA IPREIEAND IVFSVHIPLP HMEMSPWFQF MLFILQLDIA FKLNNQIREN AEVSMDVSLA YRDDAFAEWT EMAHERVPRK L KCTFTSPK TPEHEGRYYE CDVLPFMEIG SVAHKFYLLN IRLPVNEKKK INVGIGEIKD IRLVGIHQNG GFTKVWFAMK TF LTPSIFI IMVWYWRRIT MMSRPPVLLE KVIFALGISM TFINIPVEWF SIGFDWTWML LFGDIRQGIF YAMLLSFWII FCG EHMMDQ HERNHIAGYW KQVGPIAVGS FCLFIFDMCE RGVQLTNPFY SIWTTDIGTE LAMAFIIVAG ICLCLYFLFL CFMV FQVFR NISGKQSSLP AMSKVRRLHY EGLIFRFKFL MLITLACAAM TVIFFIVSQV TEGHWKWGGV TVQVNSAFFT GIYGM WNLY VFALMFLYAP SHKNYGEDQS NGDLGVHSGE ELQLTTTITH VDGPTEIYKL TRKEAQEAEN LYFQSHHHHH HHHHHD YKD DDDK

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Macromolecule #5: PALMITIC ACID

MacromoleculeName: PALMITIC ACID / type: ligand / ID: 5 / Number of copies: 1 / Formula: PLM
Molecular weightTheoretical: 256.424 Da
Chemical component information

ChemComp-PLM:
PALMITIC ACID / Palmitic acid

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Macromolecule #6: 1-CIS-9-OCTADECANOYL-2-CIS-9-HEXADECANOYL PHOSPHATIDYL GLYCEROL

MacromoleculeName: 1-CIS-9-OCTADECANOYL-2-CIS-9-HEXADECANOYL PHOSPHATIDYL GLYCEROL
type: ligand / ID: 6 / Number of copies: 3 / Formula: DR9
Molecular weightTheoretical: 746.991 Da
Chemical component information

ChemComp-DR9:
1-CIS-9-OCTADECANOYL-2-CIS-9-HEXADECANOYL PHOSPHATIDYL GLYCEROL

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Macromolecule #7: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 7 / Number of copies: 1 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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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

Concentration1 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
100.0 mMTris
150.0 mMNaClSodium chloride
GridModel: Quantifoil R0.6/1 / Material: GOLD / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Digitization - Sampling interval: 5.0 µm / Number grids imaged: 1 / Number real images: 12747 / Average exposure time: 3.0 sec. / Average electron dose: 58.0 e/Å2

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Image processing

Particle selectionNumber selected: 4415933
CTF correctionSoftware - Name: cryoSPARC (ver. 2.15) / Details: Patch CTF
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.19 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15) / Software - details: Local refinement / Number images used: 27288

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Atomic model buiding 1

Initial modelPDB ID:

4f0a

Output model

PDB-7kc4:
Human WLS in complex with WNT8A

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