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- PDB-6xbj: Structure of human SMO-D384R complex with Gi -

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Basic information

Entry
Database: PDB / ID: 6xbj
TitleStructure of human SMO-D384R complex with Gi
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 / 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 / dorsal/ventral neural tube patterning / forebrain morphogenesis / 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 / regulation of mitotic spindle organization / homeostasis of number of cells within a tissue / 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 / multicellular organism growth / Activation of the phototransduction cascade / 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 / 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 / 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 / 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
Similarity search - Domain/homology
CHOLESTEROL / 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: 3.88 Å
AuthorsQi, X. / Long, T. / 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

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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)185,9476
Polymers185,5615
Non-polymers3871
Water0
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 / Non-polymers , 3 types, 3 molecules RS

#1: Protein Smoothened homolog / SMO / Protein Gx


Mass: 71828.547 Da / Num. of mol.: 1 / Mutation: D384R
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)
#6: Chemical ChemComp-CLR / CHOLESTEROL / 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

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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: SMO-GI COMPLEX / Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
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: DARK FIELD
Image recordingElectron dose: 60 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: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.88 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 621773 / Symmetry type: POINT
RefinementResolution: 3.88→233.24 Å / Cor.coef. Fo:Fc: 0.869 / SU B: 25.461 / SU ML: 0.334 / ESU R: 0.229 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.37581 --
obs0.37581 454804 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: 212.878 Å2
Baniso -1Baniso -2Baniso -3
1-0.55 Å2-0.69 Å20.25 Å2
2--0.63 Å2-0.93 Å2
3----1.18 Å2
Refinement stepCycle: 1 / Total: 10198
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0030.01310439
ELECTRON MICROSCOPYr_bond_other_d0.0020.0179528
ELECTRON MICROSCOPYr_angle_refined_deg1.3721.64214158
ELECTRON MICROSCOPYr_angle_other_deg1.1271.5822049
ELECTRON MICROSCOPYr_dihedral_angle_1_deg7.19351292
ELECTRON MICROSCOPYr_dihedral_angle_2_deg32.49521.704534
ELECTRON MICROSCOPYr_dihedral_angle_3_deg14.482151716
ELECTRON MICROSCOPYr_dihedral_angle_4_deg10.9761569
ELECTRON MICROSCOPYr_chiral_restr0.1280.21368
ELECTRON MICROSCOPYr_gen_planes_refined0.0040.0211701
ELECTRON MICROSCOPYr_gen_planes_other0.0020.022334
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_it2.32923.125195
ELECTRON MICROSCOPYr_mcbond_other2.32923.125194
ELECTRON MICROSCOPYr_mcangle_it4.17234.6736478
ELECTRON MICROSCOPYr_mcangle_other4.17234.6736479
ELECTRON MICROSCOPYr_scbond_it1.89523.2855244
ELECTRON MICROSCOPYr_scbond_other1.89523.2855245
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other3.4534.8737681
ELECTRON MICROSCOPYr_long_range_B_refined7.3712235
ELECTRON MICROSCOPYr_long_range_B_other7.3712236
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.88→3.981 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.625 33628 -
obs--100 %

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