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- PDB-7v6y: Cryo-EM structure of Patched in lipid nanodisc - the wildtype, 3.... -

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Basic information

Entry
Database: PDB / ID: 7v6y
TitleCryo-EM structure of Patched in lipid nanodisc - the wildtype, 3.5 angstrom (re-processed with dataset of 7dzq)
ComponentsProtein patched homolog 1,Protein patched homolog 1
KeywordsMEMBRANE PROTEIN / Caveolae / Hedgehog signaling / Lipid nanodisc / Patched / Ptc1 dimer
Function / homology
Function and homology information


Ligand-receptor interactions / Activation of SMO / neural plate axis specification / cell differentiation involved in kidney development / hedgehog receptor activity / response to chlorate / cell proliferation involved in metanephros development / neural tube formation / smoothened binding / hedgehog family protein binding ...Ligand-receptor interactions / Activation of SMO / neural plate axis specification / cell differentiation involved in kidney development / hedgehog receptor activity / response to chlorate / cell proliferation involved in metanephros development / neural tube formation / smoothened binding / hedgehog family protein binding / hindlimb morphogenesis / Hedgehog 'on' state / epidermal cell fate specification / spinal cord motor neuron differentiation / prostate gland development / Hedgehog 'off' state / negative regulation of cell division / patched binding / somite development / dorsal/ventral neural tube patterning / smooth muscle tissue development / cellular response to cholesterol / pharyngeal system development / pattern specification process / mammary gland duct morphogenesis / mammary gland epithelial cell differentiation / cell fate determination / mammary gland development / commissural neuron axon guidance / metanephric collecting duct development / dorsal/ventral pattern formation / regulation of growth / embryonic limb morphogenesis / negative regulation of multicellular organism growth / cholesterol binding / branching involved in ureteric bud morphogenesis / positive regulation of epidermal cell differentiation / spermatid development / dendritic growth cone / keratinocyte proliferation / positive regulation of cholesterol efflux / epidermis development / negative regulation of osteoblast differentiation / negative regulation of keratinocyte proliferation / embryonic organ development / response to retinoic acid / axonal growth cone / heart morphogenesis / negative regulation of stem cell proliferation / response to mechanical stimulus / cyclin binding / regulation of mitotic cell cycle / liver regeneration / epithelial cell proliferation / animal organ morphogenesis / protein localization to plasma membrane / stem cell proliferation / negative regulation of smoothened signaling pathway / neural tube closure / brain development / protein processing / caveola / negative regulation of epithelial cell proliferation / apical part of cell / glucose homeostasis / response to estradiol / heparin binding / regulation of protein localization / regulation of cell population proliferation / midbody / in utero embryonic development / postsynaptic membrane / cilium / response to xenobiotic stimulus / negative regulation of cell population proliferation / negative regulation of DNA-templated transcription / protein-containing complex binding / positive regulation of DNA-templated transcription / perinuclear region of cytoplasm / negative regulation of transcription by RNA polymerase II / Golgi apparatus / signal transduction / extracellular region / zinc ion binding / plasma membrane
Similarity search - Function
Transmembrane receptor, patched / : / Sterol-sensing domain of SREBP cleavage-activation / Sterol-sensing domain (SSD) profile. / Sterol-sensing domain
Similarity search - Domain/homology
Chem-5VI / CHOLESTEROL / Protein patched homolog 1
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsLuo, Y. / Zhao, Y. / Qu, Q. / Li, D.
Funding support China, 4items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31570748 China
National Natural Science Foundation of China (NSFC)U1632127 China
Chinese Academy of SciencesXDB37020204 China
Chinese Academy of SciencesQYZDB-SSW-SMC037 China
CitationJournal: Structure / Year: 2021
Title: Cryo-EM study of patched in lipid nanodisc suggests a structural basis for its clustering in caveolae.
Authors: Yitian Luo / Guoyue Wan / Xiang Zhang / Xuan Zhou / Qiuwen Wang / Jialin Fan / Hongmin Cai / Liya Ma / Hailong Wu / Qianhui Qu / Yao Cong / Yun Zhao / Dianfan Li /
Abstract: The 12-transmembrane protein Patched (Ptc1) acts as a suppressor for Hedgehog (Hh) signaling by depleting sterols in the cytoplasmic membrane leaflet that are required for the activation of ...The 12-transmembrane protein Patched (Ptc1) acts as a suppressor for Hedgehog (Hh) signaling by depleting sterols in the cytoplasmic membrane leaflet that are required for the activation of downstream regulators. The positive modulator Hh inhibits Ptc1's transporter function by binding to Ptc1 and its co-receptors, which are locally concentrated in invaginated microdomains known as caveolae. Here, we reconstitute the mouse Ptc1 into lipid nanodiscs and determine its structure using single-particle cryoelectron microscopy. The structure is overall similar to those in amphipol and detergents but displays various conformational differences in the transmembrane region. Although most particles show monomers, we observe Ptc1 dimers with distinct interaction patterns and different membrane curvatures, some of which are reminiscent of caveolae. We find that an extramembranous "hand-shake" region rich in hydrophobic and aromatic residues mediates inter-Ptc1 interactions under different membrane curvatures. Our data provide a plausible framework for Ptc1 clustering in the highly curved caveolae.
History
DepositionAug 20, 2021Deposition site: PDBJ / Processing site: PDBJ
SupersessionSep 22, 2021ID: 7DZQ
Revision 1.0Sep 22, 2021Provider: repository / Type: Initial release
Revision 1.0Sep 22, 2021Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Sep 22, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Sep 22, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Sep 22, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Sep 22, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Sep 22, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Sep 22, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Feb 16, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first / _citation.title
Revision 1.2Oct 30, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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 / _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.3Mar 19, 2025Group: Data collection / Database references / Category: em_admin / pdbx_database_related
Item: _em_admin.last_update / _pdbx_database_related.details
Revision 1.1Mar 19, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / pdbx_database_related / Data content type: EM metadata / EM metadata
Item: _em_admin.last_update / _pdbx_database_related.details

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Assembly

Deposited unit
A: Protein patched homolog 1,Protein patched homolog 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)123,9808
Polymers121,6381
Non-polymers2,3437
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area2760 Å2
ΔGint12 kcal/mol
Surface area41000 Å2

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Components

#1: Protein Protein patched homolog 1,Protein patched homolog 1 / PTC / PTC1


Mass: 121637.617 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ptch1, Ptch / Production host: Homo sapiens (human) / References: UniProt: Q61115
#2: 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
#3: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H46O
#4: Chemical ChemComp-5VI / (2S)-2-azanyl-3-[[(2S)-3-butanoyloxy-2-dec-9-enoyloxy-propoxy]-oxidanyl-phosphoryl]oxy-propanoic acid


Mass: 481.474 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C20H36NO10P / Feature type: SUBJECT OF INVESTIGATION
#5: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestY
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

ComponentName: Ptc1 in lipid nanodisc / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 160 kDa/nm / Experimental value: YES
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mM(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)HEPES1
30.5 mMcalsium chlorideCaCl21
40.1 mMEthylenediaminetetraacetic acidEDTA1
50.2 mMtris(2-carboxyethyl)phosphineTCEP1
SpecimenConc.: 8.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: 120000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1500 nm / Calibrated defocus min: 1100 nm / Calibrated defocus max: 3000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.23 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4Gctf1.1.8CTF correction
7UCSF Chimeramodel fitting
9Cootmodel refinement
10PHENIXmodel refinement
11cryoSPARC3.2initial Euler assignment
12cryoSPARC3.2final Euler assignment
13cryoSPARC3.2classification
14cryoSPARC3.23D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1363356
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 172299 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingB value: 57.04 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6MG8

6mg8


Accession code: 6MG8 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0067549
ELECTRON MICROSCOPYf_angle_d0.81210346
ELECTRON MICROSCOPYf_dihedral_angle_d18.3061089
ELECTRON MICROSCOPYf_chiral_restr0.0491250
ELECTRON MICROSCOPYf_plane_restr0.0071283

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