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- PDB-6xbl: Structure of human SMO-Gi complex with SAG -

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Basic information

Entry
Database: PDB / ID: 6xbl
TitleStructure of human SMO-Gi complex with SAG
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Smoothened homolog
  • scFv16
KeywordsMEMBRANE PROTEIN / GPCR
Function / homology
Function and homology information


ventral midline determination / mesenchymal to epithelial transition involved in metanephric renal vesicle formation / response to inositol / regulation of heart morphogenesis / contact inhibition / negative regulation of hair follicle development / central nervous system neuron differentiation / 9+0 non-motile cilium / pancreas morphogenesis / regulation of somatic stem cell population maintenance ...ventral midline determination / mesenchymal to epithelial transition involved in metanephric renal vesicle formation / response to inositol / regulation of heart morphogenesis / contact inhibition / negative regulation of hair follicle development / central nervous system neuron differentiation / 9+0 non-motile cilium / pancreas morphogenesis / regulation of somatic stem cell population maintenance / epithelial-mesenchymal cell signaling / myoblast migration / atrial septum morphogenesis / spinal cord dorsal/ventral patterning / determination of left/right asymmetry in lateral mesoderm / left/right axis specification / ciliary tip / Activation of SMO / patched binding / forebrain morphogenesis / type B pancreatic cell development / somite development / positive regulation of organ growth / dorsal/ventral neural tube patterning / BBSome-mediated cargo-targeting to cilium / smooth muscle tissue development / cellular response to cholesterol / thalamus development / positive regulation of branching involved in ureteric bud morphogenesis / cerebellar cortex morphogenesis / mammary gland epithelial cell differentiation / dentate gyrus development / pattern specification process / commissural neuron axon guidance / dopaminergic neuron differentiation / oxysterol binding / positive regulation of multicellular organism growth / positive regulation of smoothened signaling pathway / Class B/2 (Secretin family receptors) / neural crest cell migration / cAMP-dependent protein kinase inhibitor activity / cell fate specification / negative regulation of DNA binding / anterior/posterior pattern specification / positive regulation of mesenchymal cell proliferation / ciliary membrane / midgut development / hair follicle morphogenesis / smoothened signaling pathway / negative regulation of epithelial cell differentiation / positive regulation of neuroblast proliferation / protein kinase A catalytic subunit binding / heart looping / endoplasmic reticulum-Golgi intermediate compartment / odontogenesis of dentin-containing tooth / neuroblast proliferation / vasculogenesis / Hedgehog 'off' state / negative regulation of protein phosphorylation / Adenylate cyclase inhibitory pathway / positive regulation of protein localization to cell cortex / T cell migration / D2 dopamine receptor binding / skeletal muscle fiber development / response to prostaglandin E / G protein-coupled serotonin receptor binding / adenylate cyclase-inhibiting serotonin receptor signaling pathway / adenylate cyclase regulator activity / homeostasis of number of cells within a tissue / regulation of mitotic spindle organization / centriole / protein sequestering activity / cellular response to forskolin / positive regulation of epithelial cell proliferation / epithelial cell proliferation / central nervous system development / astrocyte activation / Regulation of insulin secretion / G protein-coupled receptor binding / positive regulation of cholesterol biosynthetic process / Hedgehog 'on' state / G protein-coupled receptor activity / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / multicellular organism growth / cerebral cortex development / cilium / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G-protein beta/gamma-subunit complex binding / response to peptide hormone / Olfactory Signaling Pathway / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G protein-coupled acetylcholine receptor signaling pathway / G-protein activation / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / positive regulation of protein import into nucleus / Prostacyclin signalling through prostacyclin receptor
Similarity search - Function
Smoothened, transmembrane domain / Smoothened, cysteine-rich domain / 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 ...Smoothened, transmembrane domain / Smoothened, cysteine-rich domain / 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. / G-protein alpha subunit, group I / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / G protein alpha subunit / G-protein, gamma subunit / G-protein gamma subunit domain profile. / G-protein gamma-like domain / G-protein gamma-like domain superfamily / GGL domain / G protein gamma subunit-like motifs / GGL domain / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
CHOLESTEROL / Chem-V0S / 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 / cryo EM / Resolution: 3.96 Å
AuthorsQi, X. / Li, X.
CitationJournal: Nat Chem Biol / Year: 2020
Title: Sterols in an intramolecular channel of Smoothened mediate Hedgehog signaling.
Authors: Xiaofeng Qi / Lucas Friedberg / Ryan De Bose-Boyd / Tao Long / Xiaochun Li /
Abstract: Smoothened (SMO), a class Frizzled G protein-coupled receptor (class F GPCR), transduces the Hedgehog signal across the cell membrane. Sterols can bind to its extracellular cysteine-rich domain ...Smoothened (SMO), a class Frizzled G protein-coupled receptor (class F GPCR), transduces the Hedgehog signal across the cell membrane. Sterols can bind to its extracellular cysteine-rich domain (CRD) and to several sites in the seven transmembrane helices (7-TMs) of SMO. However, the mechanism by which sterols regulate SMO via multiple sites is unknown. Here we determined the structures of SMO-G complexes bound to the synthetic SMO agonist (SAG) and to 24(S),25-epoxycholesterol (24(S),25-EC). A novel sterol-binding site in the extracellular extension of TM6 was revealed to connect other sites in 7-TMs and CRD, forming an intramolecular sterol channel from the middle side of 7-TMs to CRD. Additional structures of two gain-of-function variants, SMO and SMO, showed that blocking the channel at its midpoints allows sterols to occupy the binding sites in 7-TMs, thereby activating SMO. These data indicate that sterol transport through the core of SMO is a major regulator of SMO-mediated signaling.
History
DepositionJun 6, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 30, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 25, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Oct 30, 2024Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature / refine
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_entry_details.has_protein_modification / _refine.ls_d_res_high / _refine.ls_d_res_low

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Structure visualization

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Structure viewerMolecule:
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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
S: scFv16
hetero molecules


Theoretical massNumber of molelcules
Total (without water)186,3957
Polymers185,5195
Non-polymers8772
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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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: 40415.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAI1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P63096
#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 37671.102 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Trichoplusia ni (cabbage looper) / 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: Trichoplusia ni (cabbage looper) / References: UniProt: P59768

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Protein / Antibody , 2 types, 2 molecules RS

#1: Protein Smoothened homolog / SMO / Protein Gx


Mass: 71786.438 Da / Num. of mol.: 1
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: Antibody scFv16


Mass: 27784.896 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Trichoplusia ni (cabbage looper)

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Non-polymers , 2 types, 2 molecules

#6: Chemical ChemComp-V0S / 3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-{[3-(pyridin-4-yl)phenyl]methyl}-1-benzothiophene-2-carboxamide


Mass: 490.059 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C28H28ClN3OS / Feature type: SUBJECT OF INVESTIGATION / Comment: agonist*YM
#7: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H46O / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationY

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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
1hSMO-Gi-SAGCOMPLEX#1-#50MULTIPLE SOURCES
2hSMOCOMPLEX#11RECOMBINANT
3GiCOMPLEX#2-#42RECOMBINANT
4scFv16COMPLEX#53RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
22Homo sapiens (human)9606
33Homo sapiens (human)9606
44Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
22Homo sapiens (human)9606
33Homo sapiens (human)9606
44Homo sapiens (human)9606
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen 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 FIELD
Image recordingElectron dose: 80 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0257 / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.96 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 550527 / Symmetry type: POINT
RefinementResolution: 3.96→3.96 Å / Cor.coef. Fo:Fc: 0.83 / SU B: 23.808 / SU ML: 0.319 / ESU R: 0.236 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.39224 --
obs0.39224 473381 100 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 135.59 Å2
Baniso -1Baniso -2Baniso -3
1-0.28 Å2-1.13 Å21.09 Å2
2--1.5 Å2-1.1 Å2
3----1.79 Å2
Refinement stepCycle: 1 / Total: 10248
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0030.01310493
ELECTRON MICROSCOPYr_bond_other_d0.0030.0179568
ELECTRON MICROSCOPYr_angle_refined_deg1.3291.64614234
ELECTRON MICROSCOPYr_angle_other_deg1.1871.58422142
ELECTRON MICROSCOPYr_dihedral_angle_1_deg7.85151295
ELECTRON MICROSCOPYr_dihedral_angle_2_deg31.32521.764533
ELECTRON MICROSCOPYr_dihedral_angle_3_deg15.181151717
ELECTRON MICROSCOPYr_dihedral_angle_4_deg13.0621568
ELECTRON MICROSCOPYr_chiral_restr0.1330.21374
ELECTRON MICROSCOPYr_gen_planes_refined0.0040.0211752
ELECTRON MICROSCOPYr_gen_planes_other0.0020.022345
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_it1.45314.7595207
ELECTRON MICROSCOPYr_mcbond_other1.45314.7595206
ELECTRON MICROSCOPYr_mcangle_it2.70122.1336493
ELECTRON MICROSCOPYr_mcangle_other2.70122.1346494
ELECTRON MICROSCOPYr_scbond_it0.92114.8045286
ELECTRON MICROSCOPYr_scbond_other0.92114.8045287
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other1.7922.1917742
ELECTRON MICROSCOPYr_long_range_B_refined5.40512193
ELECTRON MICROSCOPYr_long_range_B_other5.40512194
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.901→4.002 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.506 35278 -
obs--100 %

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