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- PDB-7v6z: Cryo-EM structure of Patched1 (V1084A mutant) in lipid nanodisc, ... -

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Basic information

Entry
Database: PDB / ID: 7v6z
TitleCryo-EM structure of Patched1 (V1084A mutant) in lipid nanodisc, 3.64 angstrom (reprocessed with the dataset of 7dzp)
ComponentsProtein patched homolog 1,Protein patched homolog 1
KeywordsMEMBRANE PROTEIN / Caveolae / Hedgehog signaling / Lipid nanodisc / Patched / Ptc1 dimer / Thermostable mutant.
Function / homology
Function and homology information


cell differentiation involved in kidney development / response to chlorate / cell proliferation involved in metanephros development / hindlimb morphogenesis / Ligand-receptor interactions / Activation of SMO / epidermal cell fate specification / neural plate axis specification / neural tube patterning / hedgehog receptor activity ...cell differentiation involved in kidney development / response to chlorate / cell proliferation involved in metanephros development / hindlimb morphogenesis / Ligand-receptor interactions / Activation of SMO / epidermal cell fate specification / neural plate axis specification / neural tube patterning / hedgehog receptor activity / smoothened binding / spinal cord motor neuron differentiation / hedgehog family protein binding / neural tube formation / negative regulation of multicellular organism growth / limb morphogenesis / Hedgehog 'on' state / mammary gland duct morphogenesis / mammary gland epithelial cell differentiation / pharyngeal system development / prostate gland development / somite development / negative regulation of cell division / Hedgehog 'off' state / patched binding / smooth muscle tissue development / pattern specification process / cell fate determination / cellular response to cholesterol / mammary gland development / embryonic limb morphogenesis / dorsal/ventral pattern formation / commissural neuron axon guidance / metanephric collecting duct development / response to alkaloid / branching involved in ureteric bud morphogenesis / regulation of growth / dorsal/ventral neural tube patterning / spermatid development / positive regulation of epidermal cell differentiation / cholesterol binding / epidermis development / keratinocyte proliferation / dendritic growth cone / embryonic organ development / heart morphogenesis / positive regulation of cholesterol efflux / response to mechanical stimulus / response to retinoic acid / negative regulation of osteoblast differentiation / neural tube closure / axonal growth cone / animal organ morphogenesis / liver regeneration / regulation of mitotic cell cycle / cyclin binding / protein localization to plasma membrane / negative regulation of smoothened signaling pathway / brain development / protein processing / negative regulation of epithelial cell proliferation / caveola / in utero embryonic development / apical part of cell / glucose homeostasis / response to estradiol / regulation of protein localization / regulation of cell population proliferation / heparin binding / midbody / cilium / postsynaptic membrane / response to xenobiotic stimulus / negative regulation of cell population proliferation / negative regulation of DNA-templated transcription / positive regulation of DNA-templated transcription / protein-containing complex binding / perinuclear region of cytoplasm / Golgi apparatus / negative regulation of transcription by RNA polymerase II / signal transduction / extracellular region / zinc ion binding / plasma membrane
Similarity search - Function
Transformer protein 2 N-terminal domain / Transmembrane receptor, patched / : / Sterol-sensing domain of SREBP cleavage-activation / Sterol-sensing domain (SSD) profile. / Sterol-sensing domain
Similarity search - Domain/homology
CHOLESTEROL / Protein patched homolog 1
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.64 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: 7DZP
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: Data collection / Database references / Category: citation / citation_author / em_imaging
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.title / _em_imaging.calibrated_defocus_max / _em_imaging.calibrated_defocus_min / _em_imaging.nominal_defocus_max / _em_imaging.nominal_defocus_min
Revision 1.2Oct 23, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _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)123,0846
Polymers121,6101
Non-polymers1,4755
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1550 Å2
ΔGint14 kcal/mol
Surface area42800 Å2

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Components

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


Mass: 121609.555 Da / Num. of mol.: 1 / Mutation: V1084A
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: 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
#4: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H46O / Feature type: SUBJECT OF INVESTIGATION
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 / Num. of real images: 5140
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4Gctf1.18CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10cryoSPARC3.2initial Euler assignment
11cryoSPARC3.2final Euler assignment
12cryoSPARC3.2classification
13cryoSPARC3.23D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 3311691
3D reconstructionResolution: 3.64 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 328720 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingB value: 57.8 / Protocol: OTHER / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.017555
ELECTRON MICROSCOPYf_angle_d0.99610369
ELECTRON MICROSCOPYf_dihedral_angle_d16.7581114
ELECTRON MICROSCOPYf_chiral_restr0.0531266
ELECTRON MICROSCOPYf_plane_restr0.0071290

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