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- EMDB-27062: Cryo-EM structure of the unliganded mSMO-PGS2 in a lipidic environment -

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Basic information

Entry
Database: EMDB / ID: EMD-27062
TitleCryo-EM structure of the unliganded mSMO-PGS2 in a lipidic environment
Map data
Sample
  • Complex: mSMO-PGS2
    • Protein or peptide: Smoothened homolog, GlgA glycogen synthase chimera
  • Ligand: CHOLESTEROL
KeywordsG-protein coupled receptor / Smoothened / unliganded state / lipid system / MEMBRANE PROTEIN
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 / contact inhibition / negative regulation of hair follicle development / negative regulation of hepatocyte proliferation / central nervous system neuron differentiation ...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 / contact inhibition / negative regulation of hair follicle development / negative regulation of hepatocyte proliferation / central nervous system neuron differentiation / 9+0 non-motile cilium / pancreas morphogenesis / regulation of somatic stem cell population maintenance / epithelial-mesenchymal cell signaling / mesenchymal to epithelial transition / myoblast migration / atrial septum morphogenesis / spinal cord dorsal/ventral patterning / Hedgehog 'on' state / axon extension involved in axon guidance / determination of left/right asymmetry in lateral mesoderm / alpha-1,4-glucan glucosyltransferase (UDP-glucose donor) activity / positive regulation of hepatic stellate cell activation / cell development / left/right axis specification / Hedgehog 'off' state / patched binding / somite development / forebrain morphogenesis / type B pancreatic cell development / thalamus development / positive regulation of organ growth / positive regulation of branching involved in ureteric bud morphogenesis / cellular response to cholesterol / dorsal/ventral neural tube patterning / smooth muscle tissue development / pattern specification process / cerebellar cortex morphogenesis / mammary gland epithelial cell differentiation / dentate gyrus development / dopaminergic neuron differentiation / commissural neuron axon guidance / oxysterol binding / positive regulation of multicellular organism growth / positive regulation of neural precursor cell proliferation / positive regulation of smoothened signaling pathway / digestive tract development / dorsal/ventral pattern formation / positive regulation of vascular associated smooth muscle cell migration / determination of left/right symmetry / neural crest cell migration / cell fate specification / cAMP-dependent protein kinase inhibitor activity / anterior/posterior pattern specification / positive regulation of mesenchymal cell proliferation / ciliary membrane / midgut development / negative regulation of epithelial cell differentiation / hair follicle morphogenesis / smoothened signaling pathway / positive regulation of neuroblast proliferation / heart looping / negative regulation of DNA binding / dendritic growth cone / protein kinase A catalytic subunit binding / odontogenesis of dentin-containing tooth / endoplasmic reticulum-Golgi intermediate compartment / protein localization to nucleus / developmental growth / hair follicle development / neuroblast proliferation / vasculogenesis / embryonic organ development / axonal growth cone / positive regulation of autophagy / heart morphogenesis / skeletal muscle fiber development / homeostasis of number of cells within a tissue / epithelial cell differentiation / protein sequestering activity / centriole / ossification / positive regulation of epithelial cell proliferation / negative regulation of protein phosphorylation / epithelial cell proliferation / central nervous system development / astrocyte activation / G protein-coupled receptor activity / multicellular organism growth / caveola / cilium / cerebral cortex development / positive regulation of protein import into nucleus / osteoblast differentiation / protein import into nucleus / endocytic vesicle membrane / late endosome / gene expression / regulation of gene expression / in utero embryonic development
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
Protein smoothened / Glycogen synthase
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsZhang K / Wu H / Hoppe N / Manglik A / Cheng Y
Funding support France, United States, 3 items
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, 2022-
Header (metadata) releaseAug 3, 2022-
Map releaseAug 3, 2022-
UpdateOct 9, 2024-
Current statusOct 9, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27062.png

Downloads & links

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Map

FileDownload / File: emd_27062.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 256 pix.
= 213.76 Å		0.84 Å/pix.
x 256 pix.
= 213.76 Å		0.84 Å/pix.
x 256 pix.
= 213.76 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.835 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0063
Minimum - Maximum-0.037992913 - 0.05168554
Average (Standard dev.)0.000017372107 (±0.0015011403)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 213.76 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_27062_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_27062_half_map_2.map
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Histogram (log scale)

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Sample components

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Entire : mSMO-PGS2

EntireName: mSMO-PGS2
Components
  • Complex: mSMO-PGS2
    • Protein or peptide: Smoothened homolog, GlgA glycogen synthase chimera
  • Ligand: CHOLESTEROL

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Supramolecule #1: mSMO-PGS2

SupramoleculeName: mSMO-PGS2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Smoothened homolog, GlgA glycogen synthase chimera

MacromoleculeName: Smoothened homolog, GlgA glycogen synthase chimera / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 80.86818 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: WSHPQFEKEN LYFQSPRLLS HCGRAAHCEP LRYNVCLGSA LPYGATTTLL AGDSDSQEEA HGKLVLWSGL RNAPRCWAVI QPLLCAVYM PKCENDRVEL PSRTLCQATR GPCAIVERER GWPDFLRCTP DHFPEGCPNE VQNIKFNSSG QCEAPLVRTD N PKSWYEDV ...String:
WSHPQFEKEN LYFQSPRLLS HCGRAAHCEP LRYNVCLGSA LPYGATTTLL AGDSDSQEEA HGKLVLWSGL RNAPRCWAVI QPLLCAVYM PKCENDRVEL PSRTLCQATR GPCAIVERER GWPDFLRCTP DHFPEGCPNE VQNIKFNSSG QCEAPLVRTD N PKSWYEDV EGCGIQCQNP LFTEAEHQDM HSYIAAFGAV TGLCTLFTLA TFVADWRNSN RYPAVILFYV NACFFVGSIG WL AQFMDGA RREIVCRADG TMRFGEPTSS ETLSCVIIFV IVYYALMAGV VWFVVLTYAW HTSFKALGTT YQPLSGKTSY FHL LTWSLP FVLTVAILAV AQVDGDSVSG ICFVGYKNYR YRAGFVLAPI GLVLIVGGYF LIRGVMTLFS IKSNHPGLLG IDCS FWNES YLTGSRDERK KSLLSKFGMD EGVTFMFIGR FDRGQKGVDV LLKAIEILSS KKEFQEMRFI IIGKGDPELE GWARS LEEK HGNVKVITEM LSREFVRELY GSVDFVIIPS YFEPFGLVAL EAMCLGAIPI ASAVGGLRDI ITNETGILVK AGDPGE LAN AILKALELSR SDLSKFRENC KKRAMSFSDQ ARMDIRLAKN ETMLRLGIFG FLAFGFVLIT FSCHFYDFFN QAEWERS FR DYVLCQANVT IGLPTKKPIP DCEIKNRPSL LVEKINLFAM FGTGIAMSTW VWTKATLLIW RRTWCRLTGH SDDEPKR

UniProtKB: Protein smoothened, Glycogen synthase, Protein smoothened

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Macromolecule #2: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 2 / Number of copies: 1 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
Sugar embeddingMaterial: nanodisc
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 68.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6o3c

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 90476
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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