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- EMDB-27063: Cryo-EM structure of the unliganded mSMO-PGS1 in a lipidic environment -

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Basic information

Entry
Database: EMDB / ID: EMD-27063
TitleCryo-EM structure of the unliganded mSMO-PGS1 in a lipidic environment
Map data
Sample
  • Complex: mSMO-PGS1
    • Protein or peptide: Smoothened, Glycogen synthase chimera
KeywordsSmoothened / unliganded state / lipid system / MEMBRANE PROTEIN
Function / homology
Function and homology information


regulation of localization / response to inositol / pancreas morphogenesis / BBSome-mediated cargo-targeting to cilium / negative regulation of hair follicle development / central nervous system neuron differentiation / ventral midline determination / mesenchymal to epithelial transition involved in metanephric renal vesicle formation / regulation of heart morphogenesis / negative regulation of epithelial cell differentiation ...regulation of localization / response to inositol / pancreas morphogenesis / BBSome-mediated cargo-targeting to cilium / negative regulation of hair follicle development / central nervous system neuron differentiation / ventral midline determination / mesenchymal to epithelial transition involved in metanephric renal vesicle formation / regulation of heart morphogenesis / negative regulation of epithelial cell differentiation / Activation of SMO / alpha-tubulin acetylation / dorsal/ventral neural tube patterning / contact inhibition / negative regulation of hepatocyte proliferation / myoblast migration / epithelial-mesenchymal cell signaling / atrial septum morphogenesis / spinal cord dorsal/ventral patterning / negative regulation of DNA binding / left/right axis specification / determination of left/right asymmetry in lateral mesoderm / axon extension involved in axon guidance / midgut development / positive regulation of hepatic stellate cell activation / cell development / Hedgehog 'on' state / mammary gland epithelial cell differentiation / type B pancreatic cell development / somite development / positive regulation of multicellular organism growth / mesenchymal to epithelial transition / positive regulation of organ growth / dentate gyrus development / patched binding / smooth muscle tissue development / Hedgehog 'off' state / pattern specification process / forebrain morphogenesis / 9+0 non-motile cilium / anterior/posterior pattern specification / cerebellar cortex morphogenesis / positive regulation of mesenchymal cell proliferation / cellular response to cholesterol / determination of left/right symmetry / positive regulation of branching involved in ureteric bud morphogenesis / thalamus development / cell fate specification / positive regulation of smoothened signaling pathway / dorsal/ventral pattern formation / oxysterol binding / neural crest cell migration / commissural neuron axon guidance / hair follicle morphogenesis / odontogenesis of dentin-containing tooth / smoothened signaling pathway / digestive tract development / homeostasis of number of cells within a tissue / positive regulation of neural precursor cell proliferation / cAMP-dependent protein kinase inhibitor activity / positive regulation of vascular associated smooth muscle cell migration / positive regulation of neuroblast proliferation / embryonic organ development / heart looping / multicellular organism growth / ciliary membrane / protein kinase A catalytic subunit binding / dendritic growth cone / endoplasmic reticulum-Golgi intermediate compartment / regulation of stem cell population maintenance / negative regulation of protein phosphorylation / vasculogenesis / heart morphogenesis / developmental growth / protein localization to nucleus / axonal growth cone / ossification / skeletal muscle fiber development / positive regulation of epithelial cell proliferation / in utero embryonic development / centriole / gene expression / astrocyte activation / positive regulation of autophagy / central nervous system development / positive regulation of protein import into nucleus / cerebral cortex development / protein sequestering activity / caveola / osteoblast differentiation / G protein-coupled receptor activity / endocytic vesicle membrane / cilium / regulation of gene expression / late endosome / negative regulation of neuron apoptotic process / postsynapse / protein stabilization / positive regulation of cell migration / negative regulation of gene expression
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.
Similarity search - Domain/homology
Biological speciesMus musculus (house mouse) / Pyrococcus abyssi (strain GE5 / Orsay) (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.7 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-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27063.png

Downloads & links

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Map

FileDownload / File: emd_27063.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.)
0.83 Å/pix.
x 256 pix.
= 213.504 Å		0.83 Å/pix.
x 256 pix.
= 213.504 Å		0.83 Å/pix.
x 256 pix.
= 213.504 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.834 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0038
Minimum - Maximum-0.013825066 - 0.024335273
Average (Standard dev.)0.000016172926 (±0.0008351533)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 213.504 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_27063_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_27063_half_map_2.map
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Sample components

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Entire : mSMO-PGS1

EntireName: mSMO-PGS1
Components
  • Complex: mSMO-PGS1
    • Protein or peptide: Smoothened, Glycogen synthase chimera

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Supramolecule #1: mSMO-PGS1

SupramoleculeName: mSMO-PGS1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Smoothened, Glycogen synthase chimera

MacromoleculeName: Smoothened, Glycogen synthase chimera / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus abyssi (strain GE5 / Orsay) (archaea) / Strain: GE5 / Orsay
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: PRLLSHCGRA AHCEPLRYNV CLGSALPYGA TTTLLAGDSD SQEEAHGKLV LWSGLRNAPR CWAVIQPLLC AVYMPKCEND RVELPSRTLC QATRGPCAIV ERERGWPDFL RCTPDHFPEG CPNEVQNIKF NSSGQCEAPL VRTDNPKSWY EDVEGCGIQC QNPLFTEAEH ...String:
PRLLSHCGRA AHCEPLRYNV CLGSALPYGA TTTLLAGDSD SQEEAHGKLV LWSGLRNAPR CWAVIQPLLC AVYMPKCEND RVELPSRTLC QATRGPCAIV ERERGWPDFL RCTPDHFPEG CPNEVQNIKF NSSGQCEAPL VRTDNPKSWY EDVEGCGIQC QNPLFTEAEH QDMHSYIAAF GAVTGLCTLF TLATFVADWR NSNRYPAVIL FYVNACFFVG SIGWLAQFMD GARREIVCRA DGTMRFGEPT SSETLSCVII FVIVYYALMA GVVWFVVLTY AWHTSFKALG TTYQPLSGKT SYFHLLTWSL PFVLTVAILA VAQVDGDSVS GICFVGYKNY RYRAGFVLAP IGLVLIVGGY FLIRGVMTLF SIKSNHPGLL GIDCSFWNES YLTGSRDERK KSLLSKFGMD EGVTFMFIGR FDRGQKGVDV LLKAIEILSS KKEFQEMRFI IIGKGDPELE GWARSLEEKH GNVKVITEML SREFVRELYG SVDFVIIPSY FEPFGLVALE AMCLGAIPIA SAVGGLRDII TNETGILVKA GDPGELANAI LKALELSRSD LSKFRENCKK RAMSFSDaas kiNETMLRLG IFGFLAFGFV LITFSCHFYD FFNQAEWERS FRDYVLCQAN VTIGLPTKKP IPDCEIKNRP SLLVEKINLF AMFGTGIAMS TWVWTKATLL IWRRTWCRLT GHSDDEPKR

UniProtKB: Protein smoothened

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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: 66.0 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: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 110330
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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