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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


regulation of localization / BBSome-mediated cargo-targeting to cilium / ventral midline determination / mesenchymal to epithelial transition involved in metanephric renal vesicle formation / response to inositol / regulation of heart morphogenesis / Activation of SMO / contact inhibition / negative regulation of hepatocyte proliferation / negative regulation of hair follicle development ...regulation of localization / BBSome-mediated cargo-targeting to cilium / ventral midline determination / mesenchymal to epithelial transition involved in metanephric renal vesicle formation / response to inositol / regulation of heart morphogenesis / Activation of SMO / contact inhibition / negative regulation of hepatocyte proliferation / negative regulation of hair follicle development / 9+0 non-motile cilium / pancreas morphogenesis / epithelial-mesenchymal cell signaling / myoblast migration / negative regulation of DNA binding / atrial septum morphogenesis / Hedgehog 'on' state / regulation of stem cell population maintenance / axon extension involved in axon guidance / spinal cord dorsal/ventral patterning / determination of left/right asymmetry in lateral mesoderm / midgut development / positive regulation of hepatic stellate cell activation / alpha-1,4-glucan glucosyltransferase (UDP-glucose donor) activity / left/right axis specification / cell development / Hedgehog 'off' state / patched binding / mesenchymal to epithelial transition / somite development / type B pancreatic cell development / forebrain morphogenesis / positive regulation of organ growth / smooth muscle tissue development / cerebellar cortex morphogenesis / mammary gland epithelial cell differentiation / cellular response to cholesterol / positive regulation of branching involved in ureteric bud morphogenesis / pattern specification process / dentate gyrus development / oxysterol binding / commissural neuron axon guidance / thalamus development / positive regulation of multicellular organism growth / positive regulation of smoothened signaling pathway / dorsal/ventral pattern formation / positive regulation of vascular associated smooth muscle cell migration / positive regulation of neural precursor cell proliferation / central nervous system neuron differentiation / determination of left/right symmetry / cAMP-dependent protein kinase inhibitor activity / cell fate specification / digestive tract development / anterior/posterior pattern specification / positive regulation of mesenchymal cell proliferation / hair follicle morphogenesis / neural crest cell migration / ciliary membrane / dorsal/ventral neural tube patterning / smoothened signaling pathway / positive regulation of neuroblast proliferation / negative regulation of epithelial cell differentiation / endoplasmic reticulum-Golgi intermediate compartment / heart looping / protein kinase A catalytic subunit binding / dendritic growth cone / odontogenesis of dentin-containing tooth / negative regulation of protein phosphorylation / developmental growth / embryonic organ development / protein localization to nucleus / vasculogenesis / axonal growth cone / heart morphogenesis / skeletal muscle fiber development / astrocyte activation / homeostasis of number of cells within a tissue / positive regulation of autophagy / ossification / positive regulation of epithelial cell proliferation / central nervous system development / centriole / protein sequestering activity / cerebral cortex development / positive regulation of protein import into nucleus / caveola / G protein-coupled receptor activity / multicellular organism growth / osteoblast differentiation / endocytic vesicle membrane / late endosome / regulation of gene expression / gene expression / in utero embryonic development / negative regulation of neuron apoptotic process / protein stabilization / postsynapse / cilium / positive regulation of cell migration / negative 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
Revision 1.2Oct 9, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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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
Water00
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


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

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 FIELD / 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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