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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 / BBSome-mediated cargo-targeting to cilium / ventral midline determination / mesenchymal to epithelial transition involved in metanephric renal vesicle formation / response to inositol / Activation of SMO / regulation of heart morphogenesis / contact inhibition / negative regulation of hair follicle development / central nervous system neuron differentiation ...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 / Activation of SMO / regulation of heart morphogenesis / contact inhibition / negative regulation of hair follicle development / central nervous system neuron differentiation / 9+0 non-motile cilium / pancreas morphogenesis / regulation of somatic stem cell population maintenance / epithelial-mesenchymal cell signaling / myoblast migration / atrial septum morphogenesis / Hedgehog 'on' state / spinal cord dorsal/ventral patterning / determination of left/right asymmetry in lateral mesoderm / cell development / left/right axis specification / Hedgehog 'off' state / patched binding / forebrain morphogenesis / type B pancreatic cell development / somite development / positive regulation of organ growth / dorsal/ventral neural tube patterning / smooth muscle tissue development / positive regulation of branching involved in ureteric bud morphogenesis / cellular response to cholesterol / thalamus development / cerebellar cortex morphogenesis / pattern specification process / mammary gland epithelial cell differentiation / dentate gyrus development / dopaminergic neuron differentiation / oxysterol binding / positive regulation of multicellular organism growth / positive regulation of smoothened signaling pathway / dorsal/ventral pattern formation / digestive tract development / determination of left/right symmetry / cell fate specification / neural crest cell migration / cAMP-dependent protein kinase inhibitor activity / negative regulation of DNA binding / anterior/posterior pattern specification / positive regulation of mesenchymal cell proliferation / ciliary membrane / hair follicle morphogenesis / midgut development / negative regulation of epithelial cell differentiation / smoothened signaling pathway / positive regulation of neuroblast proliferation / endoplasmic reticulum-Golgi intermediate compartment / protein kinase A catalytic subunit binding / heart looping / odontogenesis of dentin-containing tooth / negative regulation of protein phosphorylation / neuroblast proliferation / hair follicle development / protein localization to nucleus / developmental growth / embryonic organ development / vasculogenesis / heart morphogenesis / skeletal muscle fiber development / homeostasis of number of cells within a tissue / epithelial cell differentiation / centriole / protein sequestering activity / ossification / positive regulation of epithelial cell proliferation / central nervous system development / epithelial cell proliferation / astrocyte activation / G protein-coupled receptor activity / multicellular organism growth / cerebral cortex development / caveola / positive regulation of protein import into nucleus / protein import into nucleus / osteoblast differentiation / endocytic vesicle membrane / late endosome / regulation of gene expression / gene expression / in utero embryonic development / cell population proliferation / protein stabilization / cilium / positive regulation of cell migration / negative regulation of gene expression / negative regulation of DNA-templated transcription / apoptotic process / positive regulation of cell population proliferation / positive regulation of gene expression / negative regulation of apoptotic process / positive regulation of DNA-templated transcription
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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