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- PDB-7kc4: Human WLS in complex with WNT8A -

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Basic information

Entry
Database: PDB / ID: 7kc4
TitleHuman WLS in complex with WNT8A
Components
  • Protein Wnt-8a
  • Protein wntless homolog
KeywordsMEMBRANE PROTEIN / G-protein coupled receptor / palmitoleation / secretion / embryonic development
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
Chem-DR9 / PALMITIC ACID / CHOLESTEROL HEMISUCCINATE / Protein wntless homolog / Protein Wnt-8a
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.19 Å
AuthorsNygaard, R. / Jia, Y. / Kim, J. / Ross, D. / Parisi, G. / Clarke, O.B. / Virshup, D.M. / Mancia, F.
Funding support United States, Singapore, 2items
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, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 6, 2021Provider: repository / Type: Initial release
Revision 1.1Jan 13, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jan 20, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.year

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Structure visualization

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Assembly

Deposited unit
D: Protein Wnt-8a
B: Protein wntless homolog
hetero molecules


Theoretical massNumber of molelcules
Total (without water)110,1839
Polymers105,7022
Non-polymers4,4817
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: immunoprecipitation
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules DB

#1: Protein Protein Wnt-8a / Protein Wnt-8d


Mass: 40052.359 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: WNT8A, WNT8D / Plasmid: pEG BacMam vector / Cell line (production host): FreeStyle HEK293-F Cells / Production host: Homo sapiens (human) / References: UniProt: Q9H1J5
#2: Protein Protein wntless homolog / Integral membrane protein GPR177 / Protein evenness interrupted homolog / EVI / Putative NF-kappa-B- ...Integral membrane protein GPR177 / Protein evenness interrupted homolog / EVI / Putative NF-kappa-B-activating protein 373


Mass: 65649.430 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: WLS, C1orf139, GPR177, UNQ85/PRO18667 / Plasmid: pEG BacMam vector / Cell line (production host): FreeStyle HEK293-F cells / Production host: Homo sapiens (human) / References: UniProt: Q5T9L3

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Sugars , 2 types, 2 molecules

#3: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#4: Polysaccharide alpha-D-mannopyranose-(1-3)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D- ...alpha-D-mannopyranose-(1-3)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 1072.964 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,6,5/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3-3/a4-b1_b4-c1_c3-d1_c6-f1_d3-e1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{[(3+1)][a-D-Manp]{}}[(6+1)][a-D-Manp]{}}}}LINUCSPDB-CARE

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Non-polymers , 3 types, 5 molecules

#5: Chemical ChemComp-PLM / PALMITIC ACID / Palmitic acid


Mass: 256.424 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H32O2 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-DR9 / 1-CIS-9-OCTADECANOYL-2-CIS-9-HEXADECANOYL PHOSPHATIDYL GLYCEROL / (2R)-3-{[{[(2S)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-2-[(9E)-HEXADEC-9-ENOYLOXY]PROPYL (9E)-OCTADEC-9-ENOATE


Mass: 746.991 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C40H75O10P
#7: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H50O4

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Complex of WLS/Evi and WNT8A in nanodisc / Type: COMPLEX / Details: Nanodisc were formed using MSP1E3D1 and POPG lipid / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.1056 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Strain: FreeStyle 293-F Cells
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMTris1
2150 mMNaClSodium chloride1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: FEI TITAN
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingAverage exposure time: 3 sec. / Electron dose: 58 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 12747
Image scansSampling size: 5 µm / Width: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.19rc3_4024: / Classification: refinement
EM software
IDNameVersionCategoryDetails
4cryoSPARC2.15CTF correction
7Cootmodel fitting
12cryoSPARC2.153D reconstructionLocal refinement
13PHENIXmodel refinement
CTF correctionDetails: Patch CTF / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4415933
3D reconstructionResolution: 3.19 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 27288 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingPDB-ID: 4F0A

4f0a

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036640
ELECTRON MICROSCOPYf_angle_d0.5518949
ELECTRON MICROSCOPYf_dihedral_angle_d11.6561054
ELECTRON MICROSCOPYf_chiral_restr0.042987
ELECTRON MICROSCOPYf_plane_restr0.0031089

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