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- PDB-6ww2: Structure of human Frizzled5 by fiducial-assisted cryo-EM -

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Basic information

Entry
Database: PDB / ID: 6ww2
TitleStructure of human Frizzled5 by fiducial-assisted cryo-EM
Components
  • Frizzled-5,Soluble cytochrome b562
  • anti-BRIL Fab Heavy chain
  • anti-BRIL Fab Light chain
  • anti-Fab Nanobody
KeywordsMEMBRANE PROTEIN / Wnt pathway / Frizzled / Developmental biology
Function / homology
Function and homology information


regulation of chorionic trophoblast cell proliferation / Spemann organizer formation / embryonic camera-type eye morphogenesis / syncytiotrophoblast cell differentiation involved in labyrinthine layer development / chorionic trophoblast cell differentiation / glandular epithelial cell maturation / Signaling by RNF43 mutants / post-embryonic camera-type eye development / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / apoptotic process involved in morphogenesis ...regulation of chorionic trophoblast cell proliferation / Spemann organizer formation / embryonic camera-type eye morphogenesis / syncytiotrophoblast cell differentiation involved in labyrinthine layer development / chorionic trophoblast cell differentiation / glandular epithelial cell maturation / Signaling by RNF43 mutants / post-embryonic camera-type eye development / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / apoptotic process involved in morphogenesis / anterior/posterior axis specification, embryo / embryonic axis specification / cellular response to molecule of bacterial origin / intestinal epithelial cell maturation / Wnt receptor activity / non-canonical Wnt signaling pathway / regulation of mitophagy / eye development / Wnt-protein binding / branching involved in labyrinthine layer morphogenesis / Class B/2 (Secretin family receptors) / labyrinthine layer blood vessel development / Disassembly of the destruction complex and recruitment of AXIN to the membrane / positive regulation of protein targeting to mitochondrion / regulation of bicellular tight junction assembly / bicellular tight junction / canonical Wnt signaling pathway / synapse assembly / Regulation of FZD by ubiquitination / positive regulation of interleukin-1 beta production / Asymmetric localization of PCP proteins / clathrin-coated endocytic vesicle membrane / electron transport chain / G protein-coupled receptor activity / positive regulation of T cell cytokine production / positive regulation of type II interferon production / neuron differentiation / positive regulation of tumor necrosis factor production / amyloid-beta binding / T cell differentiation in thymus / Ca2+ pathway / early endosome membrane / angiogenesis / perikaryon / periplasmic space / electron transfer activity / iron ion binding / axon / Golgi membrane / negative regulation of cell population proliferation / synapse / lipid binding / heme binding / dendrite / ubiquitin protein ligase binding / protein-containing complex binding / protein kinase binding / perinuclear region of cytoplasm / cell surface / positive regulation of transcription by RNA polymerase II / plasma membrane
Similarity search - Function
Frizzled-5, CRD 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-5, CRD 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. / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Immunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
Soluble cytochrome b562 / Frizzled-5
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsTsutsumi, N. / Jude, K.M. / Gati, C. / Garcia, K.C.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)1R01DK115728 United States
Department of Energy (DOE, United States)DE-AC02-76SF00515 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Elife / Year: 2020
Title: Structure of human Frizzled5 by fiducial-assisted cryo-EM supports a heterodimeric mechanism of canonical Wnt signaling.
Authors: Naotaka Tsutsumi / Somnath Mukherjee / Deepa Waghray / Claudia Y Janda / Kevin M Jude / Yi Miao / John S Burg / Nanda Gowtham Aduri / Anthony A Kossiakoff / Cornelius Gati / K Christopher Garcia /
Abstract: Frizzleds (Fzd) are the primary receptors for Wnt morphogens, which are essential regulators of stem cell biology, yet the structural basis of Wnt signaling through Fzd remains poorly understood. ...Frizzleds (Fzd) are the primary receptors for Wnt morphogens, which are essential regulators of stem cell biology, yet the structural basis of Wnt signaling through Fzd remains poorly understood. Here we report the structure of an unliganded human Fzd5 determined by single-particle cryo-EM at 3.7 Å resolution, with the aid of an antibody chaperone acting as a fiducial marker. We also analyzed the topology of low-resolution XWnt8/Fzd5 complex particles, which revealed extreme flexibility between the Wnt/Fzd-CRD and the Fzd-TM regions. Analysis of Wnt/β-catenin signaling in response to Wnt3a versus a 'surrogate agonist' that cross-links Fzd to LRP6, revealed identical structure-activity relationships. Thus, canonical Wnt/β-catenin signaling appears to be principally reliant on ligand-induced Fzd/LRP6 heterodimerization, versus the allosteric mechanisms seen in structurally analogous class A G protein-coupled receptors, and Smoothened. These findings deepen our mechanistic understanding of Wnt signal transduction, and have implications for harnessing Wnt agonism in regenerative medicine.
History
DepositionMay 7, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 19, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 6, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
H: anti-BRIL Fab Heavy chain
K: anti-Fab Nanobody
L: anti-BRIL Fab Light chain
R: Frizzled-5,Soluble cytochrome b562


Theoretical massNumber of molelcules
Total (without water)135,8204
Polymers135,8204
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, +SDS PAGE
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Antibody anti-BRIL Fab Heavy chain


Mass: 24321.084 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#2: Antibody anti-Fab Nanobody


Mass: 13390.644 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#3: Antibody anti-BRIL Fab Light chain


Mass: 23353.947 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#4: Protein Frizzled-5,Soluble cytochrome b562 / hFz5 / FzE5 / Cytochrome b-562 / hFz5 / FzE5


Mass: 74754.180 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Escherichia coli (E. coli)
Gene: FZD5, C2orf31, cybC / Production host: Homo sapiens (human) / References: UniProt: Q13467, UniProt: P0ABE7
Has protein modificationY

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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

Component
IDNameTypeEntity IDParent-IDSource
1Fzd5-BRIL/Fab/Nb complexCOMPLEXall0MULTIPLE SOURCES
2anti-BRIL FabCOMPLEX#1, #31RECOMBINANT
3anti-Fab NanobodyCOMPLEX#21RECOMBINANT
4Frizzled-5COMPLEX#41RECOMBINANT
Molecular weightValue: 0.1 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human) / Cellular location: Membrane
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
12Homo sapiens (human)9606HEK293S GnTi-
23synthetic construct (others)32630
34Homo sapiens (human)9606
Natural host
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli562
23Escherichia coli562
34Homo sapiens9606
Buffer solutionpH: 7.2
Buffer component
IDConc.NameBuffer-ID
110 mMHEPES1
2150 mMSodium chloride1
30.001 % (w/v)GDN1
40.0001 % (w/v)CHS1
50.05 % (w/v)Digitonin1
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 281 K / Details: 5s blotting

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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 / Nominal magnification: 81000 X / Calibrated magnification: 81000 X / Nominal defocus max: -2000 nm / Nominal defocus min: -800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.1225 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10898
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: dev_3922: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEM3.6image acquisition
4cryoSPARC2.12.14CTF correction
7PHENIX1.17.1-3660model fitting
9cryoSPARC2.12.14initial Euler assignment
10cryoSPARC2.12.14final Euler assignment
11cryoSPARC2.12.14classification
12cryoSPARC2.12.143D reconstruction
13PHENIX1.17.1-3660model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6483398
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 207021 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.017781
ELECTRON MICROSCOPYf_angle_d1.08110590
ELECTRON MICROSCOPYf_dihedral_angle_d18.7221067
ELECTRON MICROSCOPYf_chiral_restr0.0621190
ELECTRON MICROSCOPYf_plane_restr0.0081334

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