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- EMDB-26101: Human M4 muscarinic acetylcholine receptor complex with Gi1 and t... -

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Entry
Database: EMDB / ID: EMD-26101
TitleHuman M4 muscarinic acetylcholine receptor complex with Gi1 and the agonist iperoxo and positive allosteric modulator VU0467154
Map data
Sample
  • Complex: M4 mAChR bound to agonist iperoxo and the PAM VU0467154 in complex with dominant negative Galpha-i1, Gbeta1, Ggamma2, and scFv16
    • Protein or peptide: Muscarinic acetylcholine receptor M4
    • 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: Antibody fragment scFv16
    • Protein or peptide: Guanine nucleotide-binding protein G(i) subunit alpha-1
  • Ligand: 4-(4,5-dihydro-1,2-oxazol-3-yloxy)-N,N,N-trimethylbut-2-yn-1-aminium
  • Ligand: 5-amino-3,4-dimethyl-N-{[4-(trifluoromethanesulfonyl)phenyl]methyl}thieno[2,3-c]pyridazine-6-carboxamide
Keywords7 transmembrane receptor / SIGNALING PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


Muscarinic acetylcholine receptors / G protein-coupled acetylcholine receptor activity / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / regulation of locomotion / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / adenylate cyclase inhibitor activity / positive regulation of protein localization to cell cortex / Adenylate cyclase inhibitory pathway / T cell migration / D2 dopamine receptor binding ...Muscarinic acetylcholine receptors / G protein-coupled acetylcholine receptor activity / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / regulation of locomotion / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / adenylate cyclase inhibitor activity / positive regulation of protein localization to cell cortex / Adenylate cyclase inhibitory pathway / T cell migration / D2 dopamine receptor binding / response to prostaglandin E / adenylate cyclase regulator activity / G protein-coupled serotonin receptor binding / adenylate cyclase-inhibiting serotonin receptor signaling pathway / cellular response to forskolin / regulation of mitotic spindle organization / Regulation of insulin secretion / positive regulation of cholesterol biosynthetic process / negative regulation of insulin secretion / G protein-coupled receptor binding / response to peptide hormone / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G-protein beta/gamma-subunit complex binding / centriolar satellite / Olfactory Signaling Pathway / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G protein-coupled acetylcholine receptor signaling pathway / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / G-protein activation / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through CDC42 / Glucagon signaling in metabolic regulation / G beta:gamma signalling through BTK / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / ADP signalling through P2Y purinoceptor 12 / photoreceptor disc membrane / Sensory perception of sweet, bitter, and umami (glutamate) taste / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / GDP binding / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / G alpha (z) signalling events / cellular response to catecholamine stimulus / ADP signalling through P2Y purinoceptor 1 / ADORA2B mediated anti-inflammatory cytokines production / G beta:gamma signalling through PI3Kgamma / adenylate cyclase-activating dopamine receptor signaling pathway / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / GPER1 signaling / Inactivation, recovery and regulation of the phototransduction cascade / cellular response to prostaglandin E stimulus / G-protein beta-subunit binding / heterotrimeric G-protein complex / G alpha (12/13) signalling events / sensory perception of taste / extracellular vesicle / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / retina development in camera-type eye / G protein activity / GTPase binding / Ca2+ pathway / fibroblast proliferation / midbody / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / cell cortex / G alpha (i) signalling events / G alpha (s) signalling events / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (q) signalling events / chemical synaptic transmission / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / Ras protein signal transduction / postsynaptic membrane / Extra-nuclear estrogen signaling / cell surface receptor signaling pathway / cell population proliferation / ciliary basal body / G protein-coupled receptor signaling pathway / lysosomal membrane / cell division / GTPase activity / dendrite / synapse / centrosome / GTP binding / protein-containing complex binding / nucleolus / magnesium ion binding / Golgi apparatus / signal transduction / extracellular exosome / nucleoplasm / membrane
Similarity search - Function
Muscarinic acetylcholine receptor M4 / Muscarinic acetylcholine receptor family / G-protein alpha subunit, group I / Serpentine type 7TM GPCR chemoreceptor Srsx / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / G protein alpha subunit / G-protein, gamma subunit ...Muscarinic acetylcholine receptor M4 / Muscarinic acetylcholine receptor family / G-protein alpha subunit, group I / Serpentine type 7TM GPCR chemoreceptor Srsx / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / G protein alpha subunit / G-protein, gamma subunit / G-protein gamma subunit domain profile. / G-protein gamma-like domain / G-protein gamma-like domain superfamily / GGL domain / G protein gamma subunit-like motifs / GGL domain / G protein beta WD-40 repeat protein / 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. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Muscarinic acetylcholine receptor M4 / 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(i) subunit alpha-1
Similarity search - Component
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsVuckovic Z / Mobbs JI
Funding support United Kingdom, Australia, Japan, 7 items
OrganizationGrant numberCountry
Wellcome Trust201529/Z/16/Z United Kingdom
National Health and Medical Research Council (NHMRC, Australia)1055134 Australia
National Health and Medical Research Council (NHMRC, Australia)1150083 Australia
National Health and Medical Research Council (NHMRC, Australia)1138448 Australia
Australian Research Council (ARC)DE170100152 Australia
Australian Research Council (ARC)IC200100052 Australia
Japan Science and Technology18069571 Japan
CitationJournal: Elife / Year: 2023
Title: Structural and dynamic mechanisms of allostery at the M4 muscarinic acetylcholine receptor
Authors: Thal DM
History
DepositionJan 30, 2022-
Header (metadata) releaseMay 17, 2023-
Map releaseMay 17, 2023-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26101.png

Downloads & links

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Map

FileDownload / File: emd_26101.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.
= 212.48 Å		0.83 Å/pix.
x 256 pix.
= 212.48 Å		0.83 Å/pix.
x 256 pix.
= 212.48 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0135
Minimum - Maximum-0.030583465 - 0.07177792
Average (Standard dev.)0.000044656263 (±0.002483786)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 212.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26101_msk_1.map
Projections & Slices
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Half map: #1

Fileemd_26101_half_map_1.map
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Half map: #2

Fileemd_26101_half_map_2.map
Projections & Slices
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Sample components

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Entire : M4 mAChR bound to agonist iperoxo and the PAM VU0467154 in comple...

EntireName: M4 mAChR bound to agonist iperoxo and the PAM VU0467154 in complex with dominant negative Galpha-i1, Gbeta1, Ggamma2, and scFv16
Components
  • Complex: M4 mAChR bound to agonist iperoxo and the PAM VU0467154 in complex with dominant negative Galpha-i1, Gbeta1, Ggamma2, and scFv16
    • Protein or peptide: Muscarinic acetylcholine receptor M4
    • 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: Antibody fragment scFv16
    • Protein or peptide: Guanine nucleotide-binding protein G(i) subunit alpha-1
  • Ligand: 4-(4,5-dihydro-1,2-oxazol-3-yloxy)-N,N,N-trimethylbut-2-yn-1-aminium
  • Ligand: 5-amino-3,4-dimethyl-N-{[4-(trifluoromethanesulfonyl)phenyl]methyl}thieno[2,3-c]pyridazine-6-carboxamide

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Supramolecule #1: M4 mAChR bound to agonist iperoxo and the PAM VU0467154 in comple...

SupramoleculeName: M4 mAChR bound to agonist iperoxo and the PAM VU0467154 in complex with dominant negative Galpha-i1, Gbeta1, Ggamma2, and scFv16
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Muscarinic acetylcholine receptor M4

MacromoleculeName: Muscarinic acetylcholine receptor M4 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 39.598277 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DYKDDDDAMA NFTPVNGSSG NQSVRLVTSS SHNRYETVEM VFIATVTGSL SLVTVVGNIL VMLSIKVNRQ LQTVNNYFLF SLACADLII GAFSMNLYTV YIIKGYWPLG AVVCDLWLAL DYVVSNASVM NLLIISFDRY FCVTKPLTYP ARRTTKMAGL M IAAAWVLS ...String:
DYKDDDDAMA NFTPVNGSSG NQSVRLVTSS SHNRYETVEM VFIATVTGSL SLVTVVGNIL VMLSIKVNRQ LQTVNNYFLF SLACADLII GAFSMNLYTV YIIKGYWPLG AVVCDLWLAL DYVVSNASVM NLLIISFDRY FCVTKPLTYP ARRTTKMAGL M IAAAWVLS FVLWAPAILF WQFVVGKRTV PDNQCFIQFL SNPAVTFGTA IAAFYLPVVI MTVLYIHISL ASRSRVHKHR PE GPKEKKA KTKRQMAARE RKVTRTIFAI LLAFILTWTP YNVMVLVNTF CQSCIPDTVW SIGYWLCYVN STINPACYAL CNA TFKKTF RHLLLCQYRN IGTARHHHHH HHH

UniProtKB: Muscarinic acetylcholine receptor M4, Muscarinic acetylcholine receptor M4

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Macromolecule #2: 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: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 38.402867 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: HHHHHHGSSG SELDQLRQEA EQLKNQIRDA RKACADATLS QITNNIDPVG RIQMRTRRTL RGHLAKIYAM HWGTDSRLLV SASQDGKLI IWDSYTTNKV HAIPLRSSWV MTCAYAPSGN YVACGGLDNI CSIYNLKTRE GNVRVSRELA GHTGYLSCCR F LDDNQIVT ...String:
HHHHHHGSSG SELDQLRQEA EQLKNQIRDA RKACADATLS QITNNIDPVG RIQMRTRRTL RGHLAKIYAM HWGTDSRLLV SASQDGKLI IWDSYTTNKV HAIPLRSSWV MTCAYAPSGN YVACGGLDNI CSIYNLKTRE GNVRVSRELA GHTGYLSCCR F LDDNQIVT SSGDTTCALW DIETGQQTTT FTGHTGDVMS LSLAPDTRLF VSGACDASAK LWDVREGMCR QTFTGHESDI NA ICFFPNG NAFATGSDDA TCRLFDLRAD QELMTYSHDN IICGITSVSF SKSGRLLLAG YDDFNCNVWD ALKADRAGVL AGH DNRVSC LGVTDDGMAV ATGSWDSFLK IWN

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

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Macromolecule #3: 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: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 7.729947 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
ASNNTASIAQ ARKLVEQLKM EANIDRIKVS KAAADLMAYC EAHAKEDPLL TPVPASENPF REKKFFCAIL

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

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Macromolecule #4: Antibody fragment scFv16

MacromoleculeName: Antibody fragment scFv16 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 26.466486 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
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 K

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Macromolecule #5: Guanine nucleotide-binding protein G(i) subunit alpha-1

MacromoleculeName: Guanine nucleotide-binding protein G(i) subunit alpha-1
type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 40.414047 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGCTLSAEDK AAVERSKMID RNLREDGEKA AREVKLLLLG AGESGKNTIV KQMKIIHEAG YSEEECKQYK AVVYSNTIQS IIAIIRAMG RLKIDFGDSA RADDARQLFV LAGAAEEGFM TAELAGVIKR LWKDSGVQAC FNRSREYQLN DSAAYYLNDL D RIAQPNYI ...String:
MGCTLSAEDK AAVERSKMID RNLREDGEKA AREVKLLLLG AGESGKNTIV KQMKIIHEAG YSEEECKQYK AVVYSNTIQS IIAIIRAMG RLKIDFGDSA RADDARQLFV LAGAAEEGFM TAELAGVIKR LWKDSGVQAC FNRSREYQLN DSAAYYLNDL D RIAQPNYI PTQQDVLRTR VKTTGIVETH FTFKDLHFKM FDVGAQRSER KKWIHCFEGV TAIIFCVALS DYDLVLAEDE EM NRMHASM KLFDSICNNK WFTDTSIILF LNKKDLFEEK IKKSPLTICY PEYAGSNTYE EAAAYIQCQF EDLNKRKDTK EIY THFTCS TDTKNVQFVF DAVTDVIIKN NLKDCGLF

UniProtKB: Guanine nucleotide-binding protein G(i) subunit alpha-1

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Macromolecule #6: 4-(4,5-dihydro-1,2-oxazol-3-yloxy)-N,N,N-trimethylbut-2-yn-1-aminium

MacromoleculeName: 4-(4,5-dihydro-1,2-oxazol-3-yloxy)-N,N,N-trimethylbut-2-yn-1-aminium
type: ligand / ID: 6 / Number of copies: 1 / Formula: IXO
Molecular weightTheoretical: 197.254 Da
Chemical component information

ChemComp-IXO:
4-(4,5-dihydro-1,2-oxazol-3-yloxy)-N,N,N-trimethylbut-2-yn-1-aminium

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Macromolecule #7: 5-amino-3,4-dimethyl-N-{[4-(trifluoromethanesulfonyl)phenyl]methy...

MacromoleculeName: 5-amino-3,4-dimethyl-N-{[4-(trifluoromethanesulfonyl)phenyl]methyl}thieno[2,3-c]pyridazine-6-carboxamide
type: ligand / ID: 7 / Number of copies: 1 / Formula: IUI
Molecular weightTheoretical: 444.451 Da
Chemical component information

ChemComp-IUI:
5-amino-3,4-dimethyl-N-{[4-(trifluoromethanesulfonyl)phenyl]methyl}thieno[2,3-c]pyridazine-6-carboxamide

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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

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 59.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 677392
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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