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- EMDB-41145: Cryo-EM Structure of GPR61- -

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Basic information

Entry
Database: EMDB / ID: EMD-41145
TitleCryo-EM Structure of GPR61-
Map data
Sample
  • Complex: GPR61-ICL3-BRIL chimera in complex with anti-BRIL Fab24 BAK5 and hinge-binding nanobody bound to inverse agonist Compound 1
    • Protein or peptide: G-protein coupled receptor 61,Soluble cytochrome b562
    • Protein or peptide: Fab24 BAK5 heavy chain
    • Protein or peptide: Fab hinge-binding nanobody
    • Protein or peptide: Fab24 BAK5 light chain
  • Ligand: 6-{[(3,5-difluoropyridin-4-yl)methyl]amino}-N-(4-ethoxy-6-methylpyrimidin-2-yl)-2-methoxy-N-(2-methoxyethyl)pyridine-3-sulfonamide
  • Ligand: water
KeywordsGPCR / inverse agonist / membrane protein / BRIL / SIGNALING PROTEIN
Function / homology
Function and homology information


ligand-independent adenylate cyclase-activating G protein-coupled receptor signaling pathway / arrestin family protein binding / electron transport chain / G protein-coupled receptor activity / periplasmic space / electron transfer activity / receptor complex / endosome membrane / endosome / iron ion binding ...ligand-independent adenylate cyclase-activating G protein-coupled receptor signaling pathway / arrestin family protein binding / electron transport chain / G protein-coupled receptor activity / periplasmic space / electron transfer activity / receptor complex / endosome membrane / endosome / iron ion binding / G protein-coupled receptor signaling pathway / heme binding / plasma membrane
Similarity search - Function
Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / 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)
Similarity search - Domain/homology
Soluble cytochrome b562 / G-protein coupled receptor 61
Similarity search - Component
Biological speciesHomo sapiens (human) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.94 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_41145.png

Downloads & links

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Map

FileDownload / File: emd_41145.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.18 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-2.4934075 - 3.8189194
Average (Standard dev.)-0.0011705464 (±0.060795538)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
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_41145_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_41145_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-ICL3-BRIL chimera in complex with anti-BRIL Fab24 BAK5 and ...

EntireName: GPR61-ICL3-BRIL chimera in complex with anti-BRIL Fab24 BAK5 and hinge-binding nanobody bound to inverse agonist Compound 1
Components
  • Complex: GPR61-ICL3-BRIL chimera in complex with anti-BRIL Fab24 BAK5 and hinge-binding nanobody bound to inverse agonist Compound 1
    • Protein or peptide: G-protein coupled receptor 61,Soluble cytochrome b562
    • Protein or peptide: Fab24 BAK5 heavy chain
    • Protein or peptide: Fab hinge-binding nanobody
    • Protein or peptide: Fab24 BAK5 light chain
  • Ligand: 6-{[(3,5-difluoropyridin-4-yl)methyl]amino}-N-(4-ethoxy-6-methylpyrimidin-2-yl)-2-methoxy-N-(2-methoxyethyl)pyridine-3-sulfonamide
  • Ligand: water

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Supramolecule #1: GPR61-ICL3-BRIL chimera in complex with anti-BRIL Fab24 BAK5 and ...

SupramoleculeName: GPR61-ICL3-BRIL chimera in complex with anti-BRIL Fab24 BAK5 and hinge-binding nanobody bound to inverse agonist Compound 1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: G-protein coupled receptor 61,Soluble cytochrome b562

MacromoleculeName: G-protein coupled receptor 61,Soluble cytochrome b562 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 59.991902 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MYPYDVPDYA ESSPIPQSSG NSSTLGRVPQ TPGPSTASGV PEVGLRDVAS ESVALFFMLL LDLTAVAGNA AVMAVIAKTP ALRKFVFVF HLCLVDLLAA LTLMPLAMLS SSALFDHALF GEVACRLYLF LSVCFVSLAI LSVSAINVER YYYVVHPMRY E VRMTLGLV ...String:
MYPYDVPDYA ESSPIPQSSG NSSTLGRVPQ TPGPSTASGV PEVGLRDVAS ESVALFFMLL LDLTAVAGNA AVMAVIAKTP ALRKFVFVF HLCLVDLLAA LTLMPLAMLS SSALFDHALF GEVACRLYLF LSVCFVSLAI LSVSAINVER YYYVVHPMRY E VRMTLGLV ASVLVGVWVK ALAMASVPVL GRVSWEEGAP SVPPGCSLQW SHSAYCQLFV VVFAVLYFLL PLLLILVVYC SM FRARRQL ADLEDNWETL NDNLKVIEKA DNAAQVKDAL TKMRAAALDA QKATPPKLED KSPDSPEMKD FRHGFDILVG QID DALKLA NEGKVKEAQA AAEQLKTTRN AYIQKYLERA RSTLQKEVKA AVVLLAVGGQ FLLCWLPYFS FHLYVALSAQ PIST GQVES VVTWIGYFCF TSNPFFYGCL NRQIRGELSK QFVCFFKPAP EEELRLPSRE GSIEENFLQF LQGTGCPSES WVSRP LPSP KQEPPAVDFR IPGQIAEETS EFLEQQLTSD IIMSDSYLRP AASPRLESGS DYKDDDDAK

UniProtKB: G-protein coupled receptor 61, Soluble cytochrome b562, G-protein coupled receptor 61

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Macromolecule #2: Fab24 BAK5 heavy chain

MacromoleculeName: Fab24 BAK5 heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 52.888203 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MKKNIAFLLA SMFVFSIATN AYASDIQMTQ SPSSLSASVG DRVTITCRAS QSVSSAVAWY QQKPGKAPKL LIYSASSLYS GVPSRFSGS RSGTDFTLTI SSLQPEDFAT YYCQQYLYYS LVTFGQGTKV EIKRTVAAPS VFIFPPSDSQ LKSGTASVVC L LNNFYPRE ...String:
MKKNIAFLLA SMFVFSIATN AYASDIQMTQ SPSSLSASVG DRVTITCRAS QSVSSAVAWY QQKPGKAPKL LIYSASSLYS GVPSRFSGS RSGTDFTLTI SSLQPEDFAT YYCQQYLYYS LVTFGQGTKV EIKRTVAAPS VFIFPPSDSQ LKSGTASVVC L LNNFYPRE AKVQWKVDNA LQSGNSQESV TEQDSKDSTY SLSSTLTLSK ADYEKHKVYA CEVTHQGLSS PVTKSFNRGE MK KNIAFLL ASMFVFSIAT NAYAEISEVQ LVESGGGLVQ PGGSLRLSCA ASGFNVRSFS IHWVRQAPGK GLEWVAYISS SSG STSYAD SVKGRFTISA DTSKNTAYLQ MNSLRAEDTA VYYCARWGYW PGEPWWKAFD YWGQGTLVTV SSASTKGPSV FPLA PSSKS TSGGTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNT KVDK KVEPKS

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Macromolecule #3: Fab hinge-binding nanobody

MacromoleculeName: Fab hinge-binding nanobody / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 13.276541 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSQVQLQESG GGLVQPGGSL RLSCAASGRT ISRYAMSWFR QAPGKEREFV AVARRSGDGA FYADSVQGRF TVSRDDAKTV YLQMNSLKP EDTAVYYCAI DSDTFYSGSY DYWGQGTQVT VSS

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Macromolecule #4: Fab24 BAK5 light chain

MacromoleculeName: Fab24 BAK5 light chain / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.398066 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQYLYYSLVT FGQGTKVEIK RTVAAPSVFI FPPSDSQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ ...String:
MDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQYLYYSLVT FGQGTKVEIK RTVAAPSVFI FPPSDSQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRG

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Macromolecule #5: 6-{[(3,5-difluoropyridin-4-yl)methyl]amino}-N-(4-ethoxy-6-methylp...

MacromoleculeName: 6-{[(3,5-difluoropyridin-4-yl)methyl]amino}-N-(4-ethoxy-6-methylpyrimidin-2-yl)-2-methoxy-N-(2-methoxyethyl)pyridine-3-sulfonamide
type: ligand / ID: 5 / Number of copies: 1 / Formula: ZOB
Molecular weightTheoretical: 524.541 Da
Chemical component information

ChemComp-ZOB:
6-{[(3,5-difluoropyridin-4-yl)methyl]amino}-N-(4-ethoxy-6-methylpyrimidin-2-yl)-2-methoxy-N-(2-methoxyethyl)pyridine-3-sulfonamide

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 1 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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: 2.94 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 170451
FSC plot (resolution estimation)

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