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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 / homologyTransmembrane receptor, patched / Hedgehog 'on' state / Hedgehog 'off' state / Ligand-receptor interactions / Activation of SMO / Sterol-sensing domain / Patched family / Sterol-sensing domain of SREBP cleavage-activation / Protein patched/dispatched / Sterol-sensing domain (SSD) profile. ...Transmembrane receptor, patched / Hedgehog 'on' state / Hedgehog 'off' state / Ligand-receptor interactions / Activation of SMO / Sterol-sensing domain / Patched family / Sterol-sensing domain of SREBP cleavage-activation / Protein patched/dispatched / Sterol-sensing domain (SSD) profile. / neural plate axis specification / hedgehog receptor activity / response to chlorate / cell proliferation involved in metanephros development / smoothened signaling pathway involved in dorsal/ventral neural tube patterning / cell differentiation involved in kidney development / neural tube patterning / smoothened binding / epidermal cell fate specification / hedgehog family protein binding / hindlimb morphogenesis / neural tube formation / spinal cord motor neuron differentiation / mammary gland duct morphogenesis / dorsal/ventral neural tube patterning / patched binding / limb morphogenesis / somite development / cellular response to cholesterol / negative regulation of smoothened signaling pathway / negative regulation of cell division / keratinocyte proliferation / negative regulation of multicellular organism growth / positive regulation of cholesterol efflux / dorsal/ventral pattern formation / pharyngeal system development / positive regulation of epidermal cell differentiation / mammary gland epithelial cell differentiation / cell fate determination / commissural neuron axon guidance / mammary gland development / smoothened signaling pathway / prostate gland development / regulation of smoothened signaling pathway / embryonic organ development / pattern specification process / cholesterol binding / embryonic limb morphogenesis / negative regulation of osteoblast differentiation / regulation of mitotic cell cycle / regulation of growth / epidermis development / branching involved in ureteric bud morphogenesis / negative regulation of epithelial cell proliferation / dendritic growth cone / heart morphogenesis / protein processing / axonal growth cone / cilium / cyclin binding / protein localization to plasma membrane / response to retinoic acid / regulation of protein localization / liver regeneration / response to mechanical stimulus / neural tube closure / animal organ morphogenesis / caveola / brain development / midbody / negative regulation of DNA-binding transcription factor activity / glucose homeostasis / in utero embryonic development / regulation of cell population proliferation / response to estradiol / heparin binding / postsynaptic density / protein-containing complex binding / response to drug / intracellular membrane-bounded organelle / negative regulation of cell population proliferation / negative regulation of transcription, DNA-templated / positive regulation of transcription, DNA-templated / integral component of plasma membrane / Golgi apparatus / negative regulation of transcription by RNA polymerase II / perinuclear region of cytoplasm / signal transduction / zinc ion binding / extracellular region / plasma membrane / nucleus / Protein patched homolog 1
Function and homology information
Specimen sourceMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.6 Å resolution
AuthorsZhang, Y. / Bulkley, D. / Xin, Y. / Roberts, K.J. / Asarnow, D.E. / Sharma, A. / Myers, B.R. / Cho, W. / Cheng, Y. / Beachy, P.A.
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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Sep 13, 2018 / Release: Nov 28, 2018

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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)146,1475
Polyers145,3581
Non-polymers7894
Water0
1


  • idetical with deposited unit
  • defined by author
  • Evidence: light scattering, DLS in conjunction with gel filtration
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TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)2930
ΔGint (kcal/M)-17
Surface area (Å2)41080

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Components

#1: Protein/peptide Protein patched homolog 1 / PTC1


Mass: 145357.844 Da / Num. of mol.: 1 / 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-UNL / UNKNOWN LIGAND


Mass: 197.236 Da / Num. of mol.: 4
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 / 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)Cell: HEK293 / Organism: Homo sapiens (human) / 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 / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 23 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / 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) / Number of grids imaged: 1 / Number 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 selectionDetails: Number of particles selected after rough initial 2D classification
Number of particles selected: 378828
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 245725 / Symmetry type: POINT
Atomic model buildingRef protocol: AB INITIO MODEL / Ref space: REAL
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0067054
ELECTRON MICROSCOPYf_angle_d1.0849704
ELECTRON MICROSCOPYf_dihedral_angle_d8.8094050
ELECTRON MICROSCOPYf_chiral_restr0.0591167
ELECTRON MICROSCOPYf_plane_restr0.0081240

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