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- EMDB-41144: Cryo-EM Structure of GPR61-G protein complex stabilized by scFv16 -

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Basic information

Entry
Database: EMDB / ID: EMD-41144
TitleCryo-EM Structure of GPR61-G protein complex stabilized by scFv16
Map data
Sample
  • Complex: GPR61-GalphaS-Gbeta1-Ggamma2 complex with single-chain Fv16
    • Protein or peptide: Single-chain Fv16
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
  • Protein or peptide: GPR61 fused to dominant negative G alpha S/I N18 chimera
KeywordsGPR61 / GPCR / Signaling complex / SIGNALING PROTEIN
Function / homology
Function and homology information


ligand-independent adenylate cyclase-activating G protein-coupled receptor signaling pathway / arrestin family protein binding / PKA activation in glucagon signalling / hair follicle placode formation / developmental growth / D1 dopamine receptor binding / intracellular transport / Hedgehog 'off' state / positive regulation of cAMP-mediated signaling / adenylate cyclase-activating adrenergic receptor signaling pathway ...ligand-independent adenylate cyclase-activating G protein-coupled receptor signaling pathway / arrestin family protein binding / PKA activation in glucagon signalling / hair follicle placode formation / developmental growth / D1 dopamine receptor binding / intracellular transport / Hedgehog 'off' state / positive regulation of cAMP-mediated signaling / adenylate cyclase-activating adrenergic receptor signaling pathway / activation of adenylate cyclase activity / adenylate cyclase activator activity / trans-Golgi network membrane / G protein-coupled receptor activity / Olfactory Signaling Pathway / G-protein beta/gamma-subunit complex binding / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / bone development / G-protein activation / G protein-coupled acetylcholine receptor signaling pathway / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / adenylate cyclase-activating G protein-coupled receptor signaling pathway / ADP signalling through P2Y purinoceptor 12 / G beta:gamma signalling through BTK / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / photoreceptor disc membrane / Adrenaline,noradrenaline inhibits insulin secretion / Glucagon-type ligand receptors / platelet aggregation / cognition / Vasopressin regulates renal water homeostasis via Aquaporins / positive regulation of GTPase activity / G alpha (z) signalling events / cellular response to catecholamine stimulus / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADORA2B mediated anti-inflammatory cytokines production / adenylate cyclase-activating dopamine receptor signaling pathway / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / cellular response to prostaglandin E stimulus / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / sensory perception of taste / GPER1 signaling / G-protein beta-subunit binding / heterotrimeric G-protein complex / Inactivation, recovery and regulation of the phototransduction cascade / G alpha (12/13) signalling events / extracellular vesicle / signaling receptor complex adaptor activity / sensory perception of smell / Thrombin signalling through proteinase activated receptors (PARs) / retina development in camera-type eye / GTPase binding / Ca2+ pathway / phospholipase C-activating G protein-coupled receptor signaling pathway / positive regulation of cold-induced thermogenesis / G alpha (i) signalling events / fibroblast proliferation / G alpha (s) signalling events / G alpha (q) signalling events / Ras protein signal transduction / cell population proliferation / Extra-nuclear estrogen signaling / receptor complex / endosome membrane / endosome / G protein-coupled receptor signaling pathway / lysosomal membrane / GTPase activity / synapse / protein-containing complex binding / GTP binding / signal transduction / extracellular exosome / membrane / metal ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
G-protein alpha subunit, group S / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit / G-protein, gamma subunit / G-protein gamma subunit domain profile. / GGL domain / G-protein gamma-like domain superfamily ...G-protein alpha subunit, group S / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit / G-protein, gamma subunit / G-protein gamma subunit domain profile. / GGL domain / G-protein gamma-like domain superfamily / G-protein gamma-like domain / GGL domain / G protein gamma subunit-like motifs / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / G-protein coupled receptors family 1 signature. / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family) / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / G-protein coupled receptor 61
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.47 Å
AuthorsLees JA / Dias JM / Han S
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2023
Title: An inverse agonist of orphan receptor GPR61 acts by a G protein-competitive allosteric mechanism.
Authors: Joshua A Lees / João M Dias / Francis Rajamohan / Jean-Philippe Fortin / Rebecca O'Connor / Jimmy X Kong / Emily A G Hughes / Ethan L Fisher / Jamison B Tuttle / Gabrielle Lovett / Bethany ...Authors: Joshua A Lees / João M Dias / Francis Rajamohan / Jean-Philippe Fortin / Rebecca O'Connor / Jimmy X Kong / Emily A G Hughes / Ethan L Fisher / Jamison B Tuttle / Gabrielle Lovett / Bethany L Kormos / Rayomand J Unwalla / Lei Zhang / Anne-Marie Dechert Schmitt / Dahui Zhou / Michael Moran / Kimberly A Stevens / Kimberly F Fennell / Alison E Varghese / Andrew Maxwell / Emmaline E Cote / Yuan Zhang / Seungil Han /
Abstract: GPR61 is an orphan GPCR related to biogenic amine receptors. Its association with phenotypes relating to appetite makes it of interest as a druggable target to treat disorders of metabolism and body ...GPR61 is an orphan GPCR related to biogenic amine receptors. Its association with phenotypes relating to appetite makes it of interest as a druggable target to treat disorders of metabolism and body weight, such as obesity and cachexia. To date, the lack of structural information or a known biological ligand or tool compound has hindered comprehensive efforts to study GPR61 structure and function. Here, we report a structural characterization of GPR61, in both its active-like complex with heterotrimeric G protein and in its inactive state. Moreover, we report the discovery of a potent and selective small-molecule inverse agonist against GPR61 and structural elucidation of its allosteric binding site and mode of action. These findings offer mechanistic insights into an orphan GPCR while providing both a structural framework and tool compound to support further studies of GPR61 function and modulation.
History
DepositionJun 28, 2023-
Header (metadata) releaseOct 4, 2023-
Map releaseOct 4, 2023-
UpdateOct 4, 2023-
Current statusOct 4, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41144.png

Downloads & links

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Map

FileDownload / File: emd_41144.map.gz / Format: CCP4 / Size: 476.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.59 Å
Density
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.41533074 - 0.5942459
Average (Standard dev.)-0.00012730586 (±0.009132673)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions500500500
Spacing500500500
CellA=B=C: 295.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_41144_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_41144_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : GPR61-GalphaS-Gbeta1-Ggamma2 complex with single-chain Fv16

EntireName: GPR61-GalphaS-Gbeta1-Ggamma2 complex with single-chain Fv16
Components
  • Complex: GPR61-GalphaS-Gbeta1-Ggamma2 complex with single-chain Fv16
    • Protein or peptide: Single-chain Fv16
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
    • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
  • Protein or peptide: GPR61 fused to dominant negative G alpha S/I N18 chimera

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Supramolecule #1: GPR61-GalphaS-Gbeta1-Ggamma2 complex with single-chain Fv16

SupramoleculeName: GPR61-GalphaS-Gbeta1-Ggamma2 complex with single-chain Fv16
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1, #3-#4
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Single-chain Fv16

MacromoleculeName: Single-chain Fv16 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 28.813047 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: DVQLVESGGG LVQPGGSRKL SCSASGFAFS SFGMHWVRQA PEKGLEWVAY ISSGSGTIYY ADTVKGRFTI SRDDPKNTLF LQMTSLRSE DTAMYYCVRS IYYYGSSPFD FWGQGTTLTV SSGGGGSGGG GSGGGGSDIV MTQATSSVPV TPGESVSISC R SSKSLLHS ...String:
DVQLVESGGG LVQPGGSRKL SCSASGFAFS SFGMHWVRQA PEKGLEWVAY ISSGSGTIYY ADTVKGRFTI SRDDPKNTLF LQMTSLRSE DTAMYYCVRS IYYYGSSPFD FWGQGTTLTV SSGGGGSGGG GSGGGGSDIV MTQATSSVPV TPGESVSISC R SSKSLLHS NGNTYLYWFL QRPGQSPQLL IYRMSNLASG VPDRFSGSGS GTAFTLTISR LEAEDVGVYY CMQHLEYPLT FG AGTKLEL KGSLEVLFQG PAAAHHHHHH HH

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Macromolecule #2: GPR61 fused to dominant negative G alpha S/I N18 chimera

MacromoleculeName: GPR61 fused to dominant negative G alpha S/I N18 chimera
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 111.175773 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MKTIIALSYI FCLVFADYKD DDDAKGSGAD LEDNWETLND NLKVIEKADN AAQVKDALTK MRAAALDAQK ATPPKLEDKS PDSPEMKDF RHGFDILVGQ IDDALKLANE GKVKEAQAAA EQLKTTRNAY IQKYLLEVLF QGPESSPIPQ SSGNSSTLGR V PQTPGPST ...String:
MKTIIALSYI FCLVFADYKD DDDAKGSGAD LEDNWETLND NLKVIEKADN AAQVKDALTK MRAAALDAQK ATPPKLEDKS PDSPEMKDF RHGFDILVGQ IDDALKLANE GKVKEAQAAA EQLKTTRNAY IQKYLLEVLF QGPESSPIPQ SSGNSSTLGR V PQTPGPST ASGVPEVGLR DVASESVALF FMLLLDLTAV AGNAAVMAVI AKTPALRKFV FVFHLCLVDL LAALTLMPLA ML SSSALFD HALFGEVACR LYLFLSVCFV SLAILSVSAI NVERYYYVVH PMRYEVRMTL GLVASVLVGV WVKALAMASV PVL GRVSWE EGAPSVPPGC SLQWSHSAYC QLFVVVFAVL YFLLPLLLIL VVYCSMFRVA RVAAMQHGPL PTWMETPRQR SESL SSRST MVTSSGAPQT TPHRTFGGGK AAVVLLAVGG QFLLCWLPYF SFHLYVALSA QPISTGQVES VVTWIGYFCF TSNPF FYGC LNRQIRGELS KQFVCFFKPA PEEELRLPSR EGSIEENFLQ FLQGTGCPSE SWVSRPLPSP KQEPPAVDFR IPGQIA EET SEFLEQQLTS DIIMSDSYLR PAASPRLESG SSGSSGSSEN LYFQGSGCTL SAEDKAAVER SKMIEKQLQK DKQVYRA TH RLLLLGAGES GKNTIVKQMR ILHVNGFNGE GGEEDPQAAR SNSDGEKATK VQDIKNNLKE AIETIVAAMS NLVPPVEL A NPENQFRVDY ILSVMNVPDF DFPPEFYEHA KALWEDEGVR ACYERSNEYQ LIDCAQYFLD KIDVIKQADY VPSDQDLLR CRVLTSGIFE TKFQVDKVNF HMFDVGAQRD ERRKWIQCFN DVTAIIFVVA SSSYNMVIRE DNQTNRLQAA LKLFDSIWNN KWLRDTSVI LFLNKQDLLA EKVLAGKSKI EDYFPEFARY TTPEDATPEP GEDPRVTRAK YFIRDEFLRI STASGDGRHY C YPHFTCSV DTENIRRVFN DCRDIIQRMH LRQYELL

UniProtKB: G-protein coupled receptor 61, Guanine nucleotide-binding protein G(s) subunit alpha isoforms short

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Macromolecule #3: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 37.41693 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSELDQLRQE AEQLKNQIRD ARKACADATL SQITNNIDPV GRIQMRTRRT LRGHLAKIYA MHWGTDSRLL VSASQDGKLI IWDSYTTNK VHAIPLRSSW VMTCAYAPSG NYVACGGLDN ICSIYNLKTR EGNVRVSREL AGHTGYLSCC RFLDDNQIVT S SGDTTCAL ...String:
MSELDQLRQE AEQLKNQIRD ARKACADATL SQITNNIDPV GRIQMRTRRT LRGHLAKIYA MHWGTDSRLL VSASQDGKLI IWDSYTTNK VHAIPLRSSW VMTCAYAPSG NYVACGGLDN ICSIYNLKTR EGNVRVSREL AGHTGYLSCC RFLDDNQIVT S SGDTTCAL WDIETGQQTT TFTGHTGDVM SLSLAPDTRL FVSGACDASA KLWDVREGMC RQTFTGHESD INAICFFPNG NA FATGSDD ATCRLFDLRA DQELMTYSHD NIICGITSVS FSKSGRLLLA GYDDFNCNVW DALKADRAGV LAGHDNRVSC LGV TDDGMA VATGSWDSFL KIWN

UniProtKB: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1

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Macromolecule #4: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 7.861143 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MASNNTASIA QARKLVEQLK MEANIDRIKV SKAAADLMAY CEAHAKEDPL LTPVPASENP FREKKFFCAI L

UniProtKB: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

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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: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.6 µm
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.47 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 52887
FSC plot (resolution estimation)

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