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- PDB-6ot0: Structure of human Smoothened-Gi complex -

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Entry
Database: PDB / ID: 6ot0
TitleStructure of human Smoothened-Gi complex
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Fab heavy chainFragment antigen-binding
  • Fab light chainFragment antigen-binding
  • Smoothened homolog
KeywordsSIGNALING PROTEIN / GPCR / Complex / Hedgehog signaling
Function / homology
Function and homology information


Thrombin signalling through proteinase activated receptors (PARs) / Hedgehog 'on' state / Activation of SMO / BBSome-mediated cargo-targeting to cilium / Hedgehog 'off' state / G alpha (s) signalling events / Prostacyclin signalling through prostacyclin receptor / Adrenaline,noradrenaline inhibits insulin secretion / Ca2+ pathway / G alpha (q) signalling events ...Thrombin signalling through proteinase activated receptors (PARs) / Hedgehog 'on' state / Activation of SMO / BBSome-mediated cargo-targeting to cilium / Hedgehog 'off' state / G alpha (s) signalling events / Prostacyclin signalling through prostacyclin receptor / Adrenaline,noradrenaline inhibits insulin secretion / Ca2+ pathway / G alpha (q) signalling events / G alpha (12/13) signalling events / G beta:gamma signalling through PLC beta / ADP signalling through P2Y purinoceptor 1 / G alpha (i) signalling events / G alpha (z) signalling events / Glucagon-type ligand receptors / Regulation of insulin secretion / Thromboxane signalling through TP receptor / Vasopressin regulates renal water homeostasis via Aquaporins / G beta:gamma signalling through PI3Kgamma / ADP signalling through P2Y purinoceptor 12 / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Olfactory Signaling Pathway / PLC beta mediated events / Activation of G protein gated Potassium channels / Glucagon signaling in metabolic regulation / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / Adenylate cyclase inhibitory pathway / G-protein activation / Activation of the phototransduction cascade / G beta:gamma signalling through BTK / Inactivation, recovery and regulation of the phototransduction cascade / G beta:gamma signalling through CDC42 / Class B/2 (Secretin family receptors) / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / Extra-nuclear estrogen signaling / Presynaptic function of Kainate receptors / regulation of heart morphogenesis / mesenchymal to epithelial transition involved in metanephric renal vesicle formation / detection of cell density by contact stimulus involved in contact inhibition / negative regulation of hair follicle development / smoothened signaling pathway involved in ventral spinal cord patterning / ventral midline determination / cerebellar cortex morphogenesis / central nervous system neuron differentiation / epithelial-mesenchymal cell signaling / forebrain morphogenesis / pancreas morphogenesis / positive regulation of hh target transcription factor activity / determination of left/right asymmetry in lateral mesoderm / smoothened signaling pathway involved in regulation of cerebellar granule cell precursor cell proliferation / atrial septum morphogenesis / left/right axis specification / myoblast migration / regulation of stem cell population maintenance / dentate gyrus development / negative regulation of epithelial cell differentiation / renal system development / ciliary tip / dorsal/ventral neural tube patterning / positive regulation of organ growth / regulation of cAMP-mediated signaling / patched binding / somite development / cardiac muscle cell apoptotic process / G protein-coupled serotonin receptor binding / cellular response to cholesterol / G-protein beta/gamma-subunit complex / sensory perception of taste / cellular response to catecholamine stimulus / positive regulation of protein localization to cell cortex / G protein-coupled acetylcholine receptor signaling pathway / alkylglycerophosphoethanolamine phosphodiesterase activity / positive regulation of branching involved in ureteric bud morphogenesis / protein heterotrimerization / thalamus development / type B pancreatic cell development / positive regulation of multicellular organism growth / mammary gland epithelial cell differentiation / rhodopsin mediated signaling pathway / adenylate cyclase-activating dopamine receptor signaling pathway / cell cortex region / photoreceptor outer segment membrane / positive regulation of smoothened signaling pathway / G-protein beta-subunit binding / commissural neuron axon guidance / smoothened signaling pathway / ciliary membrane / neural crest cell migration / photoreceptor disc membrane / spectrin binding / regulation of mitotic spindle organization / G-protein beta/gamma-subunit complex binding / negative regulation of synaptic transmission / cellular response to forskolin / cell fate specification / heterotrimeric G-protein complex / anterior/posterior pattern specification / Wnt signaling pathway, calcium modulating pathway / hair follicle morphogenesis
Frizzled cysteine-rich domain superfamily / P-loop containing nucleoside triphosphate hydrolase / Smoothened, transmembrane domain / G-protein gamma-like domain superfamily / WD40-repeat-containing domain superfamily / Smoothened, cysteine-rich domain / G-protein beta WD-40 repeat / WD domain, G-beta repeat / G-protein alpha subunit / GGL domain ...Frizzled cysteine-rich domain superfamily / P-loop containing nucleoside triphosphate hydrolase / Smoothened, transmembrane domain / G-protein gamma-like domain superfamily / WD40-repeat-containing domain superfamily / Smoothened, cysteine-rich domain / G-protein beta WD-40 repeat / WD domain, G-beta repeat / G-protein alpha subunit / GGL domain / Fz domain / Frizzled/Smoothened family membrane region / Trp-Asp (WD) repeats signature. / Smoothened / Frizzled domain / G-protein, gamma subunit / Frizzled/Smoothened, transmembrane domain / Guanine nucleotide binding protein (G-protein), alpha subunit / G-protein alpha subunit, group I / G-protein, beta subunit / WD40 repeat / G protein alpha subunit, helical insertion / WD40 repeat, conserved site / Frizzled/secreted frizzled-related protein / G-protein gamma-like domain / WD40/YVTN repeat-like-containing domain superfamily / Guanine nucleotide-binding protein, beta subunit / GPCR, family 2-like / WD40-repeat-containing domain / Frizzled (fz) domain profile. / G-protein gamma subunit domain profile. / Trp-Asp (WD) repeats profile. / G-protein coupled receptors family 2 profile 2. / Trp-Asp (WD) repeats circular profile. / G-alpha domain profile.
Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(i) subunit alpha-1 / Smoothened homolog
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / Resolution: 3.84 Å
AuthorsQi, X. / Li, X.
CitationJournal: Nature / Year: 2019
Title: Cryo-EM structure of oxysterol-bound human Smoothened coupled to a heterotrimeric G.
Authors: Xiaofeng Qi / Heng Liu / Bonne Thompson / Jeffrey McDonald / Cheng Zhang / Xiaochun Li /
Abstract: The oncoprotein Smoothened (SMO), a G-protein-coupled receptor (GPCR) of the Frizzled-class (class-F), transduces the Hedgehog signal from the tumour suppressor Patched-1 (PTCH1) to the ...The oncoprotein Smoothened (SMO), a G-protein-coupled receptor (GPCR) of the Frizzled-class (class-F), transduces the Hedgehog signal from the tumour suppressor Patched-1 (PTCH1) to the glioma-associated-oncogene (GLI) transcription factors, which activates the Hedgehog signalling pathway. It has remained unknown how PTCH1 modulates SMO, how SMO is stimulated to form a complex with heterotrimeric G proteins and whether G-protein coupling contributes to the activation of GLI proteins. Here we show that 24,25-epoxycholesterol, which we identify as an endogenous ligand of PTCH1, can stimulate Hedgehog signalling in cells and can trigger G-protein signalling via human SMO in vitro. We present a cryo-electron microscopy structure of human SMO bound to 24(S),25-epoxycholesterol and coupled to a heterotrimeric G protein. The structure reveals a ligand-binding site for 24(S),25-epoxycholesterol in the 7-transmembrane region, as well as a G-coupled activation mechanism of human SMO. Notably, the G protein presents a different arrangement from that of class-A GPCR-G complexes. Our work provides molecular insights into Hedgehog signal transduction and the activation of a class-F GPCR.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: May 2, 2019 / Release: Jun 12, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jun 12, 2019Structure modelrepositoryInitial release
1.1Jun 19, 2019Structure modelData collection / Database referencescitation_citation.journal_id_CSD / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

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Assembly

Deposited unit
R: Smoothened homolog
A: Guanine nucleotide-binding protein G(i) subunit alpha-1
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
G: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
L: Fab light chain
H: Fab heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)198,5777
Polymers198,1776
Non-polymers4011
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area13540 Å2
ΔGint-84 kcal/mol
Surface area70050 Å2

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Components

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Protein/peptide , 3 types, 3 molecules RLH

#1: Protein/peptide Smoothened homolog / SMO / Protein Gx


Mass: 62109.387 Da / Num. of mol.: 1 / Fragment: residues 1-555
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SMO, SMOH / Production host: Homo sapiens (human) / References: UniProt: Q99835
#5: Protein/peptide Fab light chain / Fragment antigen-binding


Mass: 23258.783 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Escherichia coli (E. coli)
#6: Protein/peptide Fab heavy chain / Fragment antigen-binding


Mass: 25788.822 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Escherichia coli (E. coli)

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Guanine nucleotide-binding protein ... , 3 types, 3 molecules ABG

#2: Protein/peptide Guanine nucleotide-binding protein G(i) subunit alpha-1 / Adenylate cyclase-inhibiting G alpha protein


Mass: 40414.047 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAI1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P63096
#3: Protein/peptide Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 38744.371 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P62873
#4: Protein/peptide Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 7861.143 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNG2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P59768

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Non-polymers , 1 types, 1 molecules

#7: Chemical ChemComp-CO1 / 17-[3-(3,3-DIMETHYL-OXIRANYL)-1-METHYL-PROPYL]-10,13-DIMETHYL-2,3,4,7,8,9,10,11,12,13,14,15,16,17-TETRADECAHYDRO-1H-CYCLOPENTA[A]PHENANTHREN-3-OL / 24,25(S)-EPOXYCHOLESTEROL


Mass: 400.637 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H44O2

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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

Source: RECOMBINANT / Type: COMPLEX

IDNameEntity IDParent-ID
1Smoothened-Gi-Fab complex1, 2, 3, 4, 5, 60
2Smoothened homolog11
3Guanine nucleotide-binding protein G(i) subunit alpha-1, Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1, Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-22, 3, 41
4Fab light chain, Fab heavy chain5, 61
Molecular weightValue: 0.250 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
34Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Spodoptera frugiperda (fall armyworm)7108
34Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: NO

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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: DARK FIELD
Image recordingElectron dose: 1.4 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0158 / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.84 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 141100 / Symmetry type: POINT
RefinementResolution: 3.9→94.36 Å / Cor.coef. Fo:Fc: 0.876 / SU B: 148.702 / SU ML: 1.573 / ESU R: 0.975
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.4328 --
Obs0.4328 73967 99.99 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 200.285 Å2
Baniso -1Baniso -2Baniso -3
1--0.83 Å20.35 Å22.16 Å2
2--1.06 Å2-0.5 Å2
3----0.22 Å2
Refinement stepCycle: 1 / Total: 10572
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealDev ideal targetNumber
r_bond_refined_d0.0080.01910809
r_bond_other_d0.0020.029886
r_angle_refined_deg1.1591.94414659
r_angle_other_deg0.999322870
r_dihedral_angle_1_deg6.72951339
r_dihedral_angle_2_deg34.65123.555481
r_dihedral_angle_3_deg15.8151798
r_dihedral_angle_4_deg13.751569
r_chiral_restr0.0660.21636
r_gen_planes_refined0.0050.0212045
r_gen_planes_other0.0020.022348
r_nbd_refined
r_nbd_other
r_nbtor_refined
r_nbtor_other
r_xyhbond_nbd_refined
r_xyhbond_nbd_other
r_metal_ion_refined
r_metal_ion_other
r_symmetry_vdw_refined
r_symmetry_vdw_other
r_symmetry_hbond_refined
r_symmetry_hbond_other
r_symmetry_metal_ion_refined
r_symmetry_metal_ion_other
r_mcbond_it2.84220.5235374
r_mcbond_other2.84220.5235373
r_mcangle_it5.24230.7746707
r_mcangle_other5.24230.7746708
r_scbond_it2.15920.5055435
r_scbond_other2.15820.5045436
r_scangle_it
r_scangle_other4.04830.7037950
r_long_range_B_refined9.84412537
r_long_range_B_other9.84412538
r_rigid_bond_restr
r_sphericity_free
r_sphericity_bonded
LS refinement shellResolution: 3.9→4.001 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.555 5502 -
Rfree-0 -
Obs--99.98 %

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