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- PDB-8cxo: Cryo-EM structure of the unliganded mSMO-PGS2 in a lipidic environment -

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Basic information

Entry
Database: PDB / ID: 8cxo
TitleCryo-EM structure of the unliganded mSMO-PGS2 in a lipidic environment
ComponentsSmoothened homolog, GlgA glycogen synthase chimera
KeywordsMEMBRANE PROTEIN / G-protein coupled receptor / Smoothened / unliganded state / lipid system
Function / homology
Function and homology information


BBSome-mediated cargo-targeting to cilium / regulation of localization / ventral midline determination / mesenchymal to epithelial transition involved in metanephric renal vesicle formation / Activation of SMO / regulation of heart morphogenesis / negative regulation of hair follicle development / negative regulation of hepatocyte proliferation / central nervous system neuron differentiation / 9+0 non-motile cilium ...BBSome-mediated cargo-targeting to cilium / regulation of localization / ventral midline determination / mesenchymal to epithelial transition involved in metanephric renal vesicle formation / Activation of SMO / regulation of heart morphogenesis / negative regulation of hair follicle development / negative regulation of hepatocyte proliferation / central nervous system neuron differentiation / 9+0 non-motile cilium / regulation of somatic stem cell population maintenance / pancreas morphogenesis / mesenchymal to epithelial transition / epithelial-mesenchymal cell signaling / : / myoblast migration / atrial septum morphogenesis / contact inhibition / determination of left/right asymmetry in lateral mesoderm / Hedgehog 'on' state / spinal cord dorsal/ventral patterning / axon extension involved in axon guidance / positive regulation of hepatic stellate cell activation / cell development / glycogen (starch) synthase activity / left/right axis specification / Hedgehog 'off' state / patched binding / thalamus development / somite development / positive regulation of branching involved in ureteric bud morphogenesis / type B pancreatic cell development / forebrain morphogenesis / cellular response to cholesterol / dorsal/ventral neural tube patterning / 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 / positive regulation of neural precursor cell proliferation / dopaminergic neuron differentiation / commissural neuron axon guidance / oxysterol binding / digestive tract development / positive regulation of smoothened signaling pathway / positive regulation of vascular associated smooth muscle cell migration / dorsal/ventral pattern formation / determination of left/right symmetry / cell fate specification / neural crest cell migration / cAMP-dependent protein kinase inhibitor activity / ciliary membrane / positive regulation of mesenchymal cell proliferation / anterior/posterior pattern specification / negative regulation of epithelial cell differentiation / midgut development / smoothened signaling pathway / hair follicle morphogenesis / positive regulation of neuroblast proliferation / heart looping / negative regulation of DNA binding / odontogenesis of dentin-containing tooth / dendritic growth cone / protein kinase A catalytic subunit binding / protein localization to nucleus / neuroblast proliferation / hair follicle development / endoplasmic reticulum-Golgi intermediate compartment / developmental growth / embryonic organ development / vasculogenesis / positive regulation of autophagy / skeletal muscle fiber development / axonal growth cone / heart morphogenesis / homeostasis of number of cells within a tissue / epithelial cell differentiation / centriole / ossification / protein sequestering activity / negative regulation of protein phosphorylation / epithelial cell proliferation / central nervous system development / positive regulation of epithelial cell proliferation / caveola / G protein-coupled receptor activity / astrocyte activation / multicellular organism growth / cilium / cerebral cortex development / osteoblast differentiation / positive regulation of protein import into nucleus / protein import into nucleus / endocytic vesicle membrane / late endosome / gene expression / regulation of gene expression
Similarity search - Function
Smoothened, transmembrane domain / Smoothened, cysteine-rich domain / Glycosyl transferases group 1 / Bacterial/plant glycogen synthase / Starch synthase, catalytic domain / Starch synthase catalytic domain / Frizzled/Smoothened, transmembrane domain / Frizzled/Smoothened family membrane region / Frizzled/Smoothened family membrane region / Frizzled/secreted frizzled-related protein ...Smoothened, transmembrane domain / Smoothened, cysteine-rich domain / Glycosyl transferases group 1 / Bacterial/plant glycogen synthase / Starch synthase, catalytic domain / Starch synthase catalytic 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.
Similarity search - Domain/homology
CHOLESTEROL / Protein smoothened / Glycogen synthase
Similarity search - Component
Biological speciesMus musculus (house mouse)
Pyrococcus abyssi (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsZhang, K. / Wu, H. / Hoppe, N. / Manglik, A. / Cheng, Y.
Funding support France, United States, 3items
OrganizationGrant numberCountry
Human Frontier Science Program (HFSP)LT000471/2017-L France
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM140847 United States
National Institutes of Health/National Center for Advancing Translational Sciences (NIH/NCATS)TR003384 United States
CitationJournal: Nat Commun / Year: 2022
Title: Fusion protein strategies for cryo-EM study of G protein-coupled receptors.
Authors: Kaihua Zhang / Hao Wu / Nicholas Hoppe / Aashish Manglik / Yifan Cheng /
Abstract: Single particle cryogenic-electron microscopy (cryo-EM) is used extensively to determine structures of activated G protein-coupled receptors (GPCRs) in complex with G proteins or arrestins. However, ...Single particle cryogenic-electron microscopy (cryo-EM) is used extensively to determine structures of activated G protein-coupled receptors (GPCRs) in complex with G proteins or arrestins. However, applying it to GPCRs without signaling proteins remains challenging because most receptors lack structural features in their soluble domains to facilitate image alignment. In GPCR crystallography, inserting a fusion protein between transmembrane helices 5 and 6 is a highly successful strategy for crystallization. Although a similar strategy has the potential to broadly facilitate cryo-EM structure determination of GPCRs alone without signaling protein, the critical determinants that make this approach successful are not yet clear. Here, we address this shortcoming by exploring different fusion protein designs, which lead to structures of antagonist bound A adenosine receptor at 3.4 Å resolution and unliganded Smoothened at 3.7 Å resolution. The fusion strategies explored here are likely applicable to cryo-EM interrogation of other GPCRs and small integral membrane proteins.
History
DepositionMay 22, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 3, 2022Provider: repository / Type: Initial release
Revision 1.1May 1, 2024Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.page_last / _citation.pdbx_database_id_PubMed / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Smoothened homolog, GlgA glycogen synthase chimera
hetero molecules


Theoretical massNumber of molelcules
Total (without water)81,2552
Polymers80,8681
Non-polymers3871
Water0
1


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

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Components

#1: Protein Smoothened homolog, GlgA glycogen synthase chimera / SMO / Glycogen synthase


Mass: 80868.180 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse), (gene. exp.) Pyrococcus abyssi (strain GE5 / Orsay) (archaea)
Gene: Smo, Smoh, PAB2292 / Strain: GE5 / Orsay / Production host: Homo sapiens (human) / References: UniProt: P56726, UniProt: Q9V2J8
#2: Chemical ChemComp-CLR / CHOLESTEROL / Cholesterol


Mass: 386.654 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H46O / Feature type: SUBJECT OF INVESTIGATION
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: mSMO-PGS2 / Type: COMPLEX / Entity ID: #1 / Source: MULTIPLE SOURCES
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: YES / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
EM embeddingMaterial: nanodisc
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 FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 68.4 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 90476 / Symmetry type: POINT

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