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- PDB-6rtx: Crystal structure of the Patched-1 (PTCH1) ectodomain 1 -

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Basic information

Entry
Database: PDB / ID: 6rtx
TitleCrystal structure of the Patched-1 (PTCH1) ectodomain 1
ComponentsProtein patched homolog 1
KeywordsMEMBRANE PROTEIN / ---- Hedgehog morphogen receptor / receptor-nanobody complex / cholesterol / palmitate / lipid-protein-modification
Function / homology
Function and homology information


cell proliferation involved in metanephros development / hedgehog receptor activity / neural plate axis specification / response to chlorate / 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 ...cell proliferation involved in metanephros development / hedgehog receptor activity / neural plate axis specification / response to chlorate / 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 / Ligand-receptor interactions / spinal cord motor neuron differentiation / limb morphogenesis / prostate gland development / Activation of SMO / negative regulation of cell division / keratinocyte proliferation / mammary gland duct morphogenesis / patched binding / somite development / cellular response to cholesterol / negative regulation of multicellular organism growth / smooth muscle tissue development / mammary gland epithelial cell differentiation / pharyngeal system development / commissural neuron axon guidance / cell fate determination / regulation of smoothened signaling pathway / Class B/2 (Secretin family receptors) / embryonic limb morphogenesis / metanephric collecting duct development / dorsal/ventral pattern formation / smoothened signaling pathway / ciliary membrane / cholesterol binding / positive regulation of epidermal cell differentiation / regulation of mitotic cell cycle / branching involved in ureteric bud morphogenesis / Hedgehog 'off' state / negative regulation of smoothened signaling pathway / dendritic growth cone / negative regulation of osteoblast differentiation / positive regulation of cholesterol efflux / heart morphogenesis / embryonic organ development / axonal growth cone / response to mechanical stimulus / response to retinoic acid / cyclin binding / protein localization to plasma membrane / caveola / regulation of protein localization / liver regeneration / Hedgehog 'on' state / neural tube closure / animal organ morphogenesis / protein processing / negative regulation of DNA-binding transcription factor activity / midbody / negative regulation of epithelial cell proliferation / glucose homeostasis / brain development / apical part of cell / response to estradiol / heparin binding / in utero embryonic development / endocytic vesicle membrane / postsynaptic density / intracellular membrane-bounded organelle / response to xenobiotic stimulus => GO:0009410 / protein-containing complex binding / negative regulation of transcription by RNA polymerase II / positive regulation of transcription, DNA-templated / perinuclear region of cytoplasm / integral component of membrane / plasma membrane / nucleus
Similarity search - Function
Transmembrane receptor, patched / Protein patched/dispatched / Patched family / Sterol-sensing domain (SSD) profile. / Sterol-sensing domain
Similarity search - Domain/homology
Protein patched homolog 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.95 Å
AuthorsRudolf, A.F. / Kowatsch, C. / El Omari, K. / Malinauskas, T. / Kinnebrew, M. / Ansell, T.B. / Bishop, B. / Pardon, E. / Schwab, R.A. / Qian, M. ...Rudolf, A.F. / Kowatsch, C. / El Omari, K. / Malinauskas, T. / Kinnebrew, M. / Ansell, T.B. / Bishop, B. / Pardon, E. / Schwab, R.A. / Qian, M. / Duman, R. / Covey, D.F. / Steyaert, J. / Wagner, A. / Sansom, M.S.P. / Rohatgi, R. / Siebold, C.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Cancer Research UKC20724/A14414 United Kingdom
Cancer Research UKC20724/A26752 United Kingdom
European Research Council647278 United Kingdom
CitationJournal: Nat Chem Biol / Year: 2019
Title: The morphogen Sonic hedgehog inhibits its receptor Patched by a pincer grasp mechanism.
Authors: Amalie F Rudolf / Maia Kinnebrew / Christiane Kowatsch / T Bertie Ansell / Kamel El Omari / Benjamin Bishop / Els Pardon / Rebekka A Schwab / Tomas Malinauskas / Mingxing Qian / Ramona Duman ...Authors: Amalie F Rudolf / Maia Kinnebrew / Christiane Kowatsch / T Bertie Ansell / Kamel El Omari / Benjamin Bishop / Els Pardon / Rebekka A Schwab / Tomas Malinauskas / Mingxing Qian / Ramona Duman / Douglas F Covey / Jan Steyaert / Armin Wagner / Mark S P Sansom / Rajat Rohatgi / Christian Siebold /
Abstract: Hedgehog (HH) ligands, classical morphogens that pattern embryonic tissues in all animals, are covalently coupled to two lipids-a palmitoyl group at the N terminus and a cholesteroyl group at the C ...Hedgehog (HH) ligands, classical morphogens that pattern embryonic tissues in all animals, are covalently coupled to two lipids-a palmitoyl group at the N terminus and a cholesteroyl group at the C terminus. While the palmitoyl group binds and inactivates Patched 1 (PTCH1), the main receptor for HH ligands, the function of the cholesterol modification has remained mysterious. Using structural and biochemical studies, along with reassessment of previous cryo-electron microscopy structures, we find that the C-terminal cholesterol attached to Sonic hedgehog (Shh) binds the first extracellular domain of PTCH1 and promotes its inactivation, thus triggering HH signaling. Molecular dynamics simulations show that this interaction leads to the closure of a tunnel through PTCH1 that serves as the putative conduit for sterol transport. Thus, Shh inactivates PTCH1 by grasping its extracellular domain with two lipidic pincers, the N-terminal palmitate and the C-terminal cholesterol, which are both inserted into the PTCH1 protein core.
History
DepositionMay 27, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 2, 2019Provider: repository / Type: Initial release
Revision 1.1Oct 9, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Jul 29, 2020Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / struct_conn / struct_site / struct_site_gen
Item: _chem_comp.name / _chem_comp.type ..._chem_comp.name / _chem_comp.type / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _struct_conn.pdbx_role
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Protein patched homolog 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)35,1133
Polymers34,6701
Non-polymers4422
Water1,78399
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area560 Å2
ΔGint7 kcal/mol
Surface area14260 Å2
MethodPISA
Unit cell
γ
α
β
Length a, b, c (Å)49.170, 65.680, 100.280
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Protein patched homolog 1 / PTC1


Mass: 34670.301 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Human PTCH1-ECD1 residues 139-428, containing three N-terminal additional residues derived from the expression vector pHLsec, and a C-terminal His6-Tag for IMAC purification
Source: (gene. exp.) Homo sapiens (human) / Gene: PTCH1, PTCH / Plasmid: pHLsec / Cell line (production host): HEK293T / Production host: Homo sapiens (human) / References: UniProt: Q13635
#2: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
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
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 99 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.37 Å3/Da / Density % sol: 48.14 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: 0.1 M bicine/Trizma base pH 8.5, 0.03M of each ethylene glycol (diethyleneglycol, triethyleneglycol, tetraethyleneglycol and pentaethyleneglycol), 12.5% (v/v) MPD, 12.5% (w/v) PEG 1000, 12.5% (w/v) PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04 / Wavelength: 0.9795 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Dec 15, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 1.95→50.14 Å / Num. obs: 24282 / % possible obs: 100 % / Redundancy: 6.4 % / CC1/2: 0.998 / Rmerge(I) obs: 0.129 / Rpim(I) all: 0.055 / Net I/σ(I): 10.7
Reflection shellResolution: 1.95→1.99 Å / Redundancy: 5.7 % / Mean I/σ(I) obs: 1.4 / Num. unique obs: 1168 / CC1/2: 0.316 / Rpim(I) all: 0.525 / % possible all: 99.1

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Processing

Software
NameVersionClassification
REFMAC5.8.0238refinement
xia2data reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6RTY
Resolution: 1.95→50.14 Å / Cor.coef. Fo:Fc: 0.958 / Cor.coef. Fo:Fc free: 0.93 / SU B: 5.484 / SU ML: 0.144 / Cross valid method: THROUGHOUT / ESU R: 0.158 / ESU R Free: 0.161 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.25696 1185 4.9 %RANDOM
Rwork0.19634 ---
obs0.19929 23043 99.89 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 36.903 Å2
Baniso -1Baniso -2Baniso -3
1-2.35 Å20 Å20 Å2
2---0.62 Å20 Å2
3----1.73 Å2
Refinement stepCycle: LAST / Resolution: 1.95→50.14 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2147 0 28 99 2274
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0080.0132243
X-RAY DIFFRACTIONr_bond_other_d0.0010.0172012
X-RAY DIFFRACTIONr_angle_refined_deg1.4291.6623054
X-RAY DIFFRACTIONr_angle_other_deg1.3271.5844704
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.4465267
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.90424.404109
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.14815382
X-RAY DIFFRACTIONr_dihedral_angle_4_deg20.576156
X-RAY DIFFRACTIONr_chiral_restr0.0650.2290
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.022459
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02435
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it2.7113.5951068
X-RAY DIFFRACTIONr_mcbond_other2.6823.5931067
X-RAY DIFFRACTIONr_mcangle_it3.985.3751332
X-RAY DIFFRACTIONr_mcangle_other3.9795.3771333
X-RAY DIFFRACTIONr_scbond_it3.9394.2651175
X-RAY DIFFRACTIONr_scbond_other3.9374.2671176
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other6.3416.1851722
X-RAY DIFFRACTIONr_long_range_B_refined8.53368.639036
X-RAY DIFFRACTIONr_long_range_B_other8.568.6118981
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.953→2.003 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.362 79 -
Rwork0.35 1666 -
obs--99.26 %

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