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

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Basic information

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


ventral midline determination / mesenchymal to epithelial transition involved in metanephric renal vesicle formation / regulation of heart morphogenesis / negative regulation of hair follicle development / 9+0 non-motile cilium / central nervous system neuron differentiation / regulation of somatic stem cell population maintenance / pancreas morphogenesis / epithelial-mesenchymal cell signaling / : ...ventral midline determination / mesenchymal to epithelial transition involved in metanephric renal vesicle formation / regulation of heart morphogenesis / negative regulation of hair follicle development / 9+0 non-motile cilium / central nervous system neuron differentiation / regulation of somatic stem cell population maintenance / pancreas morphogenesis / epithelial-mesenchymal cell signaling / : / myoblast migration / atrial septum morphogenesis / determination of left/right asymmetry in lateral mesoderm / contact inhibition / spinal cord dorsal/ventral patterning / left/right axis specification / ciliary tip / Activation of SMO / thalamus development / patched binding / somite development / positive regulation of branching involved in ureteric bud morphogenesis / type B pancreatic cell development / forebrain morphogenesis / dorsal/ventral neural tube patterning / cellular response to cholesterol / BBSome-mediated cargo-targeting to cilium / positive regulation of organ growth / smooth muscle tissue development / pattern specification process / cerebellar cortex morphogenesis / mammary gland epithelial cell differentiation / dentate gyrus development / positive regulation of multicellular organism growth / commissural neuron axon guidance / dopaminergic neuron differentiation / oxysterol binding / positive regulation of smoothened signaling pathway / Class B/2 (Secretin family receptors) / cell fate specification / neural crest cell migration / cAMP-dependent protein kinase inhibitor activity / anterior/posterior pattern specification / ciliary membrane / positive regulation of mesenchymal cell proliferation / negative regulation of epithelial cell differentiation / smoothened signaling pathway / midgut development / hair follicle morphogenesis / positive regulation of neuroblast proliferation / heart looping / negative regulation of DNA binding / odontogenesis of dentin-containing tooth / protein kinase A catalytic subunit binding / neuroblast proliferation / Adenylate cyclase inhibitory pathway / positive regulation of protein localization to cell cortex / endoplasmic reticulum-Golgi intermediate compartment / regulation of cAMP-mediated signaling / D2 dopamine receptor binding / G protein-coupled serotonin receptor binding / vasculogenesis / Hedgehog 'off' state / skeletal muscle fiber development / homeostasis of number of cells within a tissue / regulation of mitotic spindle organization / cellular response to forskolin / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / centriole / protein sequestering activity / negative regulation of protein phosphorylation / epithelial cell proliferation / central nervous system development / Regulation of insulin secretion / G protein-coupled receptor binding / positive regulation of epithelial cell proliferation / G protein-coupled receptor activity / astrocyte activation / Hedgehog 'on' state / G-protein beta/gamma-subunit complex binding / Olfactory Signaling Pathway / Activation of the phototransduction cascade / multicellular organism growth / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G-protein activation / G protein-coupled acetylcholine receptor signaling pathway / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / cilium / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / ADP signalling through P2Y purinoceptor 12 / G beta:gamma signalling through BTK / Sensory perception of sweet, bitter, and umami (glutamate) taste / cerebral cortex development / response to peptide hormone
Similarity search - Function
Smoothened, transmembrane domain / Smoothened, cysteine-rich domain / Transducin (heterotrimeric G protein), gamma chain / G Protein Gi Gamma 2 / Frizzled/Smoothened, transmembrane domain / Frizzled/Smoothened family membrane region / Frizzled/Smoothened family membrane region / Frizzled/secreted frizzled-related protein / Frizzled / Frizzled domain ...Smoothened, transmembrane domain / Smoothened, cysteine-rich domain / Transducin (heterotrimeric G protein), gamma chain / G Protein Gi Gamma 2 / Frizzled/Smoothened, transmembrane domain / Frizzled/Smoothened family membrane region / Frizzled/Smoothened family membrane region / Frizzled/secreted frizzled-related protein / Frizzled / Frizzled domain / Frizzled cysteine-rich domain superfamily / Fz domain / Frizzled (fz) domain profile. / GPCR, family 2-like / G-protein coupled receptors family 2 profile 2. / YVTN repeat-like/Quinoprotein amine dehydrogenase / G-protein alpha subunit, group I / 7 Propeller / Methylamine Dehydrogenase; Chain H / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit / G-protein, gamma subunit / G-protein gamma subunit domain profile. / GGL domain / G-protein gamma-like domain superfamily / G-protein gamma-like domain / GGL domain / G protein gamma subunit-like motifs / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / Few Secondary Structures / Irregular / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase / Mainly Beta
Similarity search - Domain/homology
Chem-CO1 / 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 / Protein smoothened
Similarity search - Component
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 glioma- ...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.
History
DepositionMay 2, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 12, 2019Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_id_CSD / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jul 24, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Dec 18, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB

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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


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area13540 Å2
ΔGint-84 kcal/mol
Surface area70050 Å2

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Components

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

#2: Protein 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 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 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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Antibody , 2 types, 2 molecules LH

#5: Antibody 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: Antibody 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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Protein / Non-polymers , 2 types, 2 molecules R

#1: Protein 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
#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
IDNameTypeEntity IDParent-IDSource
1Smoothened-Gi-Fab complexCOMPLEX#1-#60RECOMBINANT
2Smoothened homologCOMPLEX#11RECOMBINANT
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-2COMPLEX#2-#41RECOMBINANT
4Fab light chain, Fab heavy chainCOMPLEX#5-#61RECOMBINANT
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
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0080.01910809
ELECTRON MICROSCOPYr_bond_other_d0.0020.029886
ELECTRON MICROSCOPYr_angle_refined_deg1.1591.94414659
ELECTRON MICROSCOPYr_angle_other_deg0.999322870
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.72951339
ELECTRON MICROSCOPYr_dihedral_angle_2_deg34.65123.555481
ELECTRON MICROSCOPYr_dihedral_angle_3_deg15.8151798
ELECTRON MICROSCOPYr_dihedral_angle_4_deg13.751569
ELECTRON MICROSCOPYr_chiral_restr0.0660.21636
ELECTRON MICROSCOPYr_gen_planes_refined0.0050.0212045
ELECTRON MICROSCOPYr_gen_planes_other0.0020.022348
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it2.84220.5235374
ELECTRON MICROSCOPYr_mcbond_other2.84220.5235373
ELECTRON MICROSCOPYr_mcangle_it5.24230.7746707
ELECTRON MICROSCOPYr_mcangle_other5.24230.7746708
ELECTRON MICROSCOPYr_scbond_it2.15920.5055435
ELECTRON MICROSCOPYr_scbond_other2.15820.5045436
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other4.04830.7037950
ELECTRON MICROSCOPYr_long_range_B_refined9.84412537
ELECTRON MICROSCOPYr_long_range_B_other9.84412538
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_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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