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- PDB-6mg8: Structural basis for cholesterol transport-like activity of the H... -

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Basic information

Entry
Database: PDB / ID: 6mg8
TitleStructural basis for cholesterol transport-like activity of the Hedgehog receptor Patched
ComponentsProtein patched homolog 1
KeywordsMEMBRANE PROTEIN / Receptor Membrane protein
Function / homology
Function and homology information


Ligand-receptor interactions / Activation of SMO / neural plate axis specification / response to chlorate / cell differentiation involved in kidney development / hedgehog receptor activity / 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 / response to chlorate / cell differentiation involved in kidney development / hedgehog receptor activity / 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 / patched binding / negative regulation of cell division / somite development / smooth muscle tissue development / dorsal/ventral neural tube patterning / pharyngeal system development / cellular response to cholesterol / mammary gland duct morphogenesis / pattern specification process / mammary gland epithelial cell differentiation / cell fate determination / commissural neuron axon guidance / metanephric collecting duct development / mammary gland development / dorsal/ventral pattern formation / regulation of growth / embryonic limb morphogenesis / negative regulation of multicellular organism growth / branching involved in ureteric bud morphogenesis / cholesterol binding / positive regulation of epidermal cell differentiation / dendritic growth cone / keratinocyte proliferation / spermatid development / epidermis development / positive regulation of cholesterol efflux / negative regulation of keratinocyte proliferation / embryonic organ development / response to retinoic acid / negative regulation of osteoblast differentiation / response to mechanical stimulus / axonal growth cone / heart morphogenesis / negative regulation of stem cell proliferation / liver regeneration / cyclin binding / regulation of mitotic cell cycle / animal organ morphogenesis / epithelial cell proliferation / stem cell proliferation / protein localization to plasma membrane / negative regulation of smoothened signaling pathway / neural tube closure / brain development / caveola / protein processing / negative regulation of epithelial cell proliferation / apical part of cell / response to estradiol / glucose homeostasis / heparin binding / regulation of cell population proliferation / regulation of protein localization / midbody / in utero embryonic development / postsynaptic membrane / cilium / 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 / negative regulation of transcription by RNA polymerase II / Golgi apparatus / signal transduction / extracellular region / zinc ion binding / plasma membrane
Similarity search - Function
Patched SSD / 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: 3.6 Å
AuthorsZhang, Y. / Bulkley, D. / Xin, Y. / Roberts, K.J. / Asarnow, D.E. / Sharma, A. / Myers, B.R. / Cho, W. / Cheng, Y. / Beachy, P.A.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Center for Research Resources (NIH/NCRR)R01GM102498 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)R01GM098672 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)S10OD020054 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)S10OD021741 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)R35GM122530 United States
CitationJournal: Cell / Year: 2018
Title: Structural Basis for Cholesterol Transport-like Activity of the Hedgehog Receptor Patched.
Authors: Yunxiao Zhang / David P Bulkley / Yao Xin / Kelsey J Roberts / Daniel E Asarnow / Ashutosh Sharma / Benjamin R Myers / Wonhwa Cho / Yifan Cheng / Philip A Beachy /
Abstract: Hedgehog protein signals mediate tissue patterning and maintenance by binding to and inactivating their common receptor Patched, a 12-transmembrane protein that otherwise would suppress the activity ...Hedgehog protein signals mediate tissue patterning and maintenance by binding to and inactivating their common receptor Patched, a 12-transmembrane protein that otherwise would suppress the activity of the 7-transmembrane protein Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. A central hydrophobic conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Cholesterol activity in the inner leaflet of the plasma membrane is reduced by PTCH1 expression but rapidly restored by Hedgehog stimulation, suggesting that PTCH1 regulates Smoothened by controlling cholesterol availability.
History
DepositionSep 13, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 28, 2018Provider: repository / Type: Initial release
Revision 2.0Jun 9, 2021Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Derived calculations / Non-polymer description / Refinement description / Source and taxonomy / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / entity_src_gen / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_sheet_hbond / pdbx_validate_close_contact / pdbx_validate_torsion / refine / refine_ls_restr / struct_conf / struct_conn / struct_mon_prot_cis / struct_sheet_range / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.pdbx_formal_charge / _atom_site.type_symbol / _chem_comp.formula / _chem_comp.formula_weight / _chem_comp.id / _chem_comp.mon_nstd_flag / _chem_comp.name / _chem_comp.type / _entity.formula_weight / _entity.pdbx_description / _entity_src_gen.pdbx_gene_src_gene / _pdbx_entity_nonpoly.comp_id / _pdbx_entity_nonpoly.name / _pdbx_nonpoly_scheme.auth_mon_id / _pdbx_nonpoly_scheme.mon_id / _pdbx_nonpoly_scheme.pdb_mon_id / _pdbx_struct_sheet_hbond.range_1_auth_comp_id / _pdbx_struct_sheet_hbond.range_1_auth_seq_id / _pdbx_struct_sheet_hbond.range_1_label_comp_id / _pdbx_struct_sheet_hbond.range_1_label_seq_id / _pdbx_struct_sheet_hbond.range_2_auth_comp_id / _pdbx_struct_sheet_hbond.range_2_auth_seq_id / _pdbx_struct_sheet_hbond.range_2_label_comp_id / _pdbx_struct_sheet_hbond.range_2_label_seq_id / _refine.B_iso_mean / _refine.pdbx_stereochemistry_target_values / _refine_ls_restr.dev_ideal / _refine_ls_restr.number / _refine_ls_restr.type / _struct_conf.beg_auth_comp_id / _struct_conf.beg_auth_seq_id / _struct_conf.beg_label_comp_id / _struct_conf.beg_label_seq_id / _struct_conf.end_auth_comp_id / _struct_conf.end_auth_seq_id / _struct_conf.end_label_comp_id / _struct_conf.end_label_seq_id / _struct_conf.pdbx_PDB_helix_class / _struct_conf.pdbx_PDB_helix_length / _struct_conn.pdbx_dist_value / _struct_mon_prot_cis.pdbx_omega_angle / _struct_sheet_range.beg_auth_comp_id / _struct_sheet_range.beg_auth_seq_id / _struct_sheet_range.beg_label_comp_id / _struct_sheet_range.beg_label_seq_id / _struct_sheet_range.end_auth_comp_id / _struct_sheet_range.end_auth_seq_id / _struct_sheet_range.end_label_comp_id / _struct_sheet_range.end_label_seq_id
Provider: author / Type: Coordinate replacement
Revision 2.1Mar 16, 2022Group: Author supporting evidence / Database references / Category: database_2 / pdbx_audit_support
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_audit_support.funding_organization
Revision 2.2Nov 6, 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

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Structure visualization

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Assembly

Deposited unit
A: Protein patched homolog 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)146,9045
Polymers145,3581
Non-polymers1,5474
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: light scattering, DLS in conjunction with gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area2930 Å2
ΔGint-17 kcal/mol
Surface area41080 Å2

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Components

#1: Protein Protein patched homolog 1 / PTC1


Mass: 145357.844 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ptch1, Ptch / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q61115
#2: Chemical
ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C27H46O
Has protein modificationY
Nonpolymer detailsTHE AUTHORS STATE THAT THE EXACT IDENTITY OF THE LIGAND IS UNKNOWN. CHOLESTEROL WAS MODELED INTO ...THE AUTHORS STATE THAT THE EXACT IDENTITY OF THE LIGAND IS UNKNOWN. CHOLESTEROL WAS MODELED INTO THE CHOLESTEROL-LIKE DENSITY PRESENT IN THE MAP.

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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: Patched1 protein solubilized in amphipol / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.1452 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293 / Plasmid: BacMam
Buffer solutionpH: 7.4
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 23 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 / Cs: 2.7 mm
Image recordingAverage exposure time: 8 sec. / Electron dose: 38 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5236
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: dev_2608: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4GctfCTF correction
7Cootmodel fitting
9cryoSPARC1initial Euler assignment
10RELION2final Euler assignment
11RELION2classification
12RELION23D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 378828
Details: Number of particles selected after rough initial 2D classification
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 245725 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 91.48 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00367221
ELECTRON MICROSCOPYf_angle_d0.69159957
ELECTRON MICROSCOPYf_chiral_restr0.04171214
ELECTRON MICROSCOPYf_plane_restr0.00411246
ELECTRON MICROSCOPYf_dihedral_angle_d13.00974343

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