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- PDB-6rvd: Revised cryo-EM structure of the human 2:1 Ptch1-Shh complex -

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Basic information

Entry
Database: PDB / ID: 6rvd
TitleRevised cryo-EM structure of the human 2:1 Ptch1-Shh complex
Components
  • Protein patched homolog 1
  • Sonic hedgehog protein
KeywordsMEMBRANE PROTEIN / ---- Hedgehog morphogen receptor / receptor-nanobody complex / cholesterol / palmitate / lipid-protein-modification
Function / homology
Function and homology information


positive regulation of skeletal muscle cell proliferation / neural plate axis specification / right lung development / left lung development / primary prostatic bud elongation / regulation of mesenchymal cell proliferation involved in prostate gland development / mesenchymal smoothened signaling pathway involved in prostate gland development / positive regulation of sclerotome development / tracheoesophageal septum formation / negative regulation of ureter smooth muscle cell differentiation ...positive regulation of skeletal muscle cell proliferation / neural plate axis specification / right lung development / left lung development / primary prostatic bud elongation / regulation of mesenchymal cell proliferation involved in prostate gland development / mesenchymal smoothened signaling pathway involved in prostate gland development / positive regulation of sclerotome development / tracheoesophageal septum formation / negative regulation of ureter smooth muscle cell differentiation / positive regulation of ureter smooth muscle cell differentiation / negative regulation of kidney smooth muscle cell differentiation / positive regulation of kidney smooth muscle cell differentiation / morphogen activity / regulation of odontogenesis / cell differentiation involved in kidney development / positive regulation of mesenchymal cell proliferation involved in ureter development / polarity specification of anterior/posterior axis / trunk neural crest cell migration / hedgehog receptor activity / response to chlorate / neural tube patterning / Formation of lateral plate mesoderm / cell proliferation involved in metanephros development / hindgut morphogenesis / striated muscle tissue development / negative regulation of alpha-beta T cell differentiation / regulation of glial cell proliferation / regulation of prostatic bud formation / metanephric mesenchymal cell proliferation involved in metanephros development / smoothened binding / formation of anatomical boundary / lung epithelium development / positive regulation of striated muscle cell differentiation / ventral midline development / hedgehog family protein binding / trachea morphogenesis / cholesterol-protein transferase activity / bud outgrowth involved in lung branching / HHAT G278V doesn't palmitoylate Hh-Np / telencephalon regionalization / epithelial-mesenchymal cell signaling / laminin-1 binding / Ligand-receptor interactions / hindlimb morphogenesis / negative regulation of cholesterol efflux / salivary gland cavitation / spinal cord dorsal/ventral patterning / determination of left/right asymmetry in lateral mesoderm / negative regulation of mesenchymal cell apoptotic process / cell development / positive regulation of cerebellar granule cell precursor proliferation / negative regulation of T cell differentiation in thymus / epidermal cell fate specification / spinal cord motor neuron differentiation / positive regulation of T cell differentiation in thymus / cerebellar granule cell precursor proliferation / intermediate filament organization / mesenchymal cell apoptotic process / prostate gland development / embryonic skeletal system development / limb bud formation / lung lobe morphogenesis / Activation of SMO / establishment of epithelial cell polarity / skeletal muscle fiber differentiation / limb morphogenesis / thalamus development / embryonic digestive tract morphogenesis / somite development / patched binding / embryonic foregut morphogenesis / negative regulation of cell division / epithelial cell proliferation involved in salivary gland morphogenesis / hindbrain development / animal organ formation / ectoderm development / positive regulation of skeletal muscle tissue development / neuron fate commitment / stem cell development / cellular response to cholesterol / negative regulation of dopaminergic neuron differentiation / mesenchymal cell proliferation involved in lung development / skeletal muscle cell proliferation / negative thymic T cell selection / lymphoid progenitor cell differentiation / positive regulation of immature T cell proliferation in thymus / dorsal/ventral neural tube patterning / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / smooth muscle tissue development / regulation of stem cell proliferation / oligodendrocyte development / male genitalia development / artery development / positive regulation of astrocyte differentiation / pattern specification process / self proteolysis / pharyngeal system development / epithelial cell proliferation involved in prostate gland development / mammary gland epithelial cell differentiation
Similarity search - Function
Transmembrane receptor, patched / Hedgehog, N-terminal signalling domain / Hedgehog protein / Hedgehog protein, Hint domain / : / Hint module / Hedgehog amino-terminal signalling domain / Muramoyl-pentapeptide Carboxypeptidase; domain 2 - #10 / Muramoyl-pentapeptide Carboxypeptidase; domain 2 / Hedgehog signalling/DD-peptidase zinc-binding domain superfamily ...Transmembrane receptor, patched / Hedgehog, N-terminal signalling domain / Hedgehog protein / Hedgehog protein, Hint domain / : / Hint module / Hedgehog amino-terminal signalling domain / Muramoyl-pentapeptide Carboxypeptidase; domain 2 - #10 / Muramoyl-pentapeptide Carboxypeptidase; domain 2 / Hedgehog signalling/DD-peptidase zinc-binding domain superfamily / Sterol-sensing domain of SREBP cleavage-activation / Protein patched/dispatched / Patched family / Sterol-sensing domain (SSD) profile. / Sterol-sensing domain / Hint domain C-terminal / Hint (Hedgehog/Intein) domain C-terminal region / Intein N-terminal splicing region / Intein N-terminal splicing motif profile. / Hint domain N-terminal / Hint (Hedgehog/Intein) domain N-terminal region / Hint domain superfamily / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
CHOLESTEROL / PALMITIC ACID / Protein patched homolog 1 / Sonic hedgehog protein
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsEl Omari, K. / Rudolf, A.F. / Kowatsch, C. / Malinauskas, T. / Kinnebrew, M. / Ansell, T.B. / Bishop, B. / Pardon, E. / Schwab, R.A. / Qian, M. ...El Omari, K. / Rudolf, A.F. / Kowatsch, C. / 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 31, 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 1.2Dec 18, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / pdbx_struct_conn_angle / struct_asym / struct_conn / 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_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _chem_comp.name / _chem_comp.type / _pdbx_entity_nonpoly.entity_id / _pdbx_entity_nonpoly.name / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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Structure visualization

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Assembly

Deposited unit
A: Protein patched homolog 1
B: Protein patched homolog 1
C: Sonic hedgehog protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)344,94419
Polymers341,0233
Non-polymers3,92116
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area12940 Å2
ΔGint-47 kcal/mol
Surface area89810 Å2
MethodPISA

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Components

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Protein , 2 types, 3 molecules ABC

#1: Protein Protein patched homolog 1 / PTC1


Mass: 160714.406 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PTCH1, PTCH / Production host: Homo sapiens (human) / References: UniProt: Q13635
#2: Protein Sonic hedgehog protein / SHH / HHG-1 / Shh unprocessed N-terminal signaling and C-terminal autoprocessing domains / ShhNC


Mass: 19594.039 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Human SHH N-terminal signalling domain, N-terminally palmitoylated and C-terminally cholesterolylated.
Source: (gene. exp.) Homo sapiens (human) / Gene: SHH / Production host: Homo sapiens (human) / References: UniProt: Q15465

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Sugars , 2 types, 8 molecules

#3: 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-Glycam 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
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#5: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 7
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

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Non-polymers , 4 types, 8 molecules

#4: Chemical
ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C27H46O / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-PLM / PALMITIC ACID


Mass: 256.424 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H32O2 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION

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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: 2:1 PTCH1:SHH complex / Type: COMPLEX / Details: 2:1 Patched-1 (PTCH1):SHH complex / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Details: 20 mM Hepes pH 7.5, 150 mM NaCl, 0.06% Digitonin, 1mM CaCl2.
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
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: OTHER
Electron lensMode: DARK FIELD
Image recordingElectron dose: 1.6 e/Å2 / Film or detector model: GATAN K2 BASE (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1RELIONparticle selection
4CTFFINDCTF correction
7Coot0.9.9.1model fitting
13PHENIX1.14model refinementRealSpaceRefine
CTF correctionType: NONE
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 77712 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Details: This is a re-refinement of pdb entry 6E1H, also using the high-resolution crystal structures of the isolated PTCH1-ECD1 (pdb 6RTW) and PTCH1-ECD2 (pdb 6RVC).
Atomic model building
IDPDB-ID 3D fitting-ID
16E1H

6e1h

1
26RTW

6rtw

1
36RVC

6rvc

1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00917607
ELECTRON MICROSCOPYf_angle_d1.16623952
ELECTRON MICROSCOPYf_dihedral_angle_d11.4710415
ELECTRON MICROSCOPYf_chiral_restr0.0642743
ELECTRON MICROSCOPYf_plane_restr0.0072988

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