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- PDB-6rmg: Structure of PTCH1 bound to a modified Hedgehog ligand ShhN-C24II -

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Basic information

Entry
Database: PDB / ID: 6rmg
TitleStructure of PTCH1 bound to a modified Hedgehog ligand ShhN-C24II
Components
  • Protein patched homolog 1,GFP-like fluorescent chromoprotein FP506, related
  • Sonic hedgehog proteinSonic hedgehog
KeywordsMEMBRANE PROTEIN / Patched / PTCH1 / Hedgehog / ShhN
Function / homology
Function and homology information


Class B/2 (Secretin family receptors) / Hedgehog ligand biogenesis / Hh mutants that don't undergo autocatalytic processing are degraded by ERAD / Hedgehog 'off' state / Ligand-receptor interactions / Hedgehog 'on' state / Activation of SMO / HHAT G278V abrogates palmitoylation of Hh-Np / Release of Hh-Np from the secreting cell / regulation of nodal signaling pathway involved in determination of lateral mesoderm left/right asymmetry ...Class B/2 (Secretin family receptors) / Hedgehog ligand biogenesis / Hh mutants that don't undergo autocatalytic processing are degraded by ERAD / Hedgehog 'off' state / Ligand-receptor interactions / Hedgehog 'on' state / Activation of SMO / HHAT G278V abrogates palmitoylation of Hh-Np / Release of Hh-Np from the secreting cell / regulation of nodal signaling pathway involved in determination of lateral mesoderm left/right asymmetry / morphogen activity / right lung development / regulation of mesenchymal cell proliferation involved in prostate gland development / mesenchymal smoothened signaling pathway involved in prostate gland development / left lung development / positive regulation of sclerotome development / positive regulation of skeletal muscle cell proliferation / primary prostatic bud elongation / epithelial-mesenchymal signaling involved in prostate gland development / positive regulation of ureter smooth muscle cell differentiation / negative regulation of ureter smooth muscle cell differentiation / positive regulation of kidney smooth muscle cell differentiation / tracheoesophageal septum formation / negative regulation of kidney smooth muscle cell differentiation / regulation of odontogenesis / positive regulation of mesenchymal cell proliferation involved in ureter development / regulation of prostatic bud formation / neural plate axis specification / spinal cord dorsal/ventral patterning / hedgehog receptor activity / cell proliferation involved in metanephros development / cell differentiation involved in kidney development / smoothened signaling pathway involved in dorsal/ventral neural tube patterning / polarity specification of anterior/posterior axis / hindgut morphogenesis / response to chlorate / cerebellar granule cell precursor proliferation / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / striated muscle tissue development / neural tube patterning / positive regulation of immature T cell proliferation in thymus / bud outgrowth involved in lung branching / smoothened binding / metanephric mesenchymal cell proliferation involved in metanephros development / positive regulation of striated muscle cell differentiation / negative regulation of alpha-beta T cell differentiation / lung epithelium development / epidermal cell fate specification / artery development / determination of left/right asymmetry in lateral mesoderm / positive regulation of hh target transcription factor activity / ventral midline development / hedgehog family protein binding / formation of anatomical boundary / smoothened signaling pathway involved in regulation of cerebellar granule cell precursor cell proliferation / trachea morphogenesis / hindlimb morphogenesis / telencephalon regionalization / laminin-1 binding / stem cell development / negative regulation of mesenchymal cell apoptotic process / myotube differentiation / spinal cord motor neuron differentiation / salivary gland cavitation / intermediate filament organization / positive regulation of T cell differentiation in thymus / negative regulation of cholesterol efflux / neuroblast proliferation / mammary gland duct morphogenesis / renal system development / dorsal/ventral neural tube patterning / limb morphogenesis / negative regulation of transcription elongation from RNA polymerase II promoter / patched binding / cellular response to cholesterol / somite development / embryonic digestive tract morphogenesis / keratinocyte proliferation / lung lobe morphogenesis / negative regulation of cell division / embryonic foregut morphogenesis / limb bud formation / negative regulation of smoothened signaling pathway / lymphoid progenitor cell differentiation / epithelial cell proliferation involved in salivary gland morphogenesis / negative thymic T cell selection / hindbrain development / negative regulation of multicellular organism growth / dorsal/ventral pattern formation / pharyngeal system development / mesenchymal cell proliferation involved in lung development / animal organ formation / negative regulation of dopaminergic neuron differentiation / male genitalia development / thalamus development / ectoderm development / positive regulation of skeletal muscle tissue development / positive regulation of epidermal cell differentiation / mammary gland epithelial cell differentiation / positive regulation of cholesterol efflux
Sterol-sensing domain (SSD) profile. / Hedgehog protein, Hint domain / Intein N-terminal splicing motif profile. / Hedgehog, N-terminal signalling domain / Sterol-sensing domain / Green fluorescent protein, GFP / Hedgehog protein / Hedgehog amino-terminal signalling domain / Green fluorescent protein / Green fluorescent protein ...Sterol-sensing domain (SSD) profile. / Hedgehog protein, Hint domain / Intein N-terminal splicing motif profile. / Hedgehog, N-terminal signalling domain / Sterol-sensing domain / Green fluorescent protein, GFP / Hedgehog protein / Hedgehog amino-terminal signalling domain / Green fluorescent protein / Green fluorescent protein / Patched family / Hint domain superfamily / Hedgehog signalling/DD-peptidase zinc-binding domain superfamily / Green fluorescent protein-related / Intein N-terminal splicing region / Transmembrane receptor, patched / Hint domain N-terminal / Hint domain C-terminal / Protein patched/dispatched / Hint module
Protein patched homolog 1 / Sonic hedgehog protein / GFP-like fluorescent chromoprotein FP506, related
Biological speciesHomo sapiens (human)
Eimeria acervulina (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsKorkhov, V.M. / Qi, C.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation150665 Switzerland
CitationJournal: Sci Adv / Year: 2019
Title: Structural basis of sterol recognition by human hedgehog receptor PTCH1.
Authors: Chao Qi / Giulio Di Minin / Irene Vercellino / Anton Wutz / Volodymyr M Korkhov /
Abstract: Hedgehog signaling is central in embryonic development and tissue regeneration. Disruption of the pathway is linked to genetic diseases and cancer. Binding of the secreted ligand, Sonic hedgehog ...Hedgehog signaling is central in embryonic development and tissue regeneration. Disruption of the pathway is linked to genetic diseases and cancer. Binding of the secreted ligand, Sonic hedgehog (ShhN) to its receptor Patched (PTCH1) activates the signaling pathway. Here, we describe a 3.4-Å cryo-EM structure of the human PTCH1 bound to ShhN, a modified hedgehog ligand mimicking its palmitoylated form. The membrane-embedded part of PTCH1 is surrounded by 10 sterol molecules at the inner and outer lipid bilayer portion of the protein. The annular sterols interact at multiple sites with both the sterol-sensing domain (SSD) and the SSD-like domain (SSDL), which are located on opposite sides of PTCH1. The structure reveals a possible route for sterol translocation across the lipid bilayer by PTCH1 and homologous transporters.
Validation Report
SummaryFull reportAbout validation report
History
DepositionMay 6, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 9, 2019Provider: repository / Type: Initial release

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Assembly

Deposited unit
A: Protein patched homolog 1,GFP-like fluorescent chromoprotein FP506, related
B: Sonic hedgehog protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)203,21221
Polymers196,2442
Non-polymers6,96819
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area4000 Å2
ΔGint-19 kcal/mol
Surface area52220 Å2
MethodPISA

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Components

#1: Protein/peptide Protein patched homolog 1,GFP-like fluorescent chromoprotein FP506, related / PTC1


Mass: 163846.734 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Eimeria acervulina (eukaryote)
Gene: PTCH1, PTCH, EAH_00062270 / Plasmid: pACMV / Cell line (production host): HEK293S GnTI- / Production host: Homo sapiens (human) / References: UniProt: Q13635, UniProt: U6GSR1
#2: Protein/peptide Sonic hedgehog protein / Sonic hedgehog / SHH / HHG-1 / Shh unprocessed N-terminal signaling and C-terminal autoprocessing domains / ShhNC


Mass: 32397.508 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SHH / Plasmid: pET28a / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: Q15465
#3: Chemical
ChemComp-NAG / N-ACETYL-D-GLUCOSAMINE


Mass: 221.208 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C8H15NO6 / N-Acetylglucosamine
#4: Chemical
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: C31H50O4
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Zinc

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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

Type: COMPLEX

IDNameEntity IDParent-IDSource
1Complex of PTCH1 with a modified Hedgehog ligand ShhN-C24II1, 20MULTIPLE SOURCES
2Protein patched homolog 1 + GFP11RECOMBINANT
3Sonic hedgehog proteinSonic hedgehog21RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Eimeria acervulina (eukaryote)5801
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
12Homo sapiens (human)9606HEK293S GnTI-
23Escherichia coli BL21 (bacteria)511693
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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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 FIELDBright-field microscopy / Nominal defocus max: -2400 nm / Nominal defocus min: -800 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 44.7 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategory
1RELIONparticle selection
2SerialEMimage acquisition
4GctfCTF correction
7Coot0.8.9.1model fitting
9RELIONinitial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13PHENIX1.11.1-2575model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 707620
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 200679 / Num. of class averages: 1 / Symmetry type: POINT

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