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- PDB-9ejz: Human M5 muscarinic acetylcholine receptor complex with mini-Gq, ... -

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Basic information

Entry
Database: PDB / ID: 9ejz
TitleHuman M5 muscarinic acetylcholine receptor complex with mini-Gq, agonist acetylcholine and positive allosteric modulator VU6007678
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Human M5 muscarinic acetylcholine receptor
  • nanobody 35
  • scFv16
KeywordsMEMBRANE PROTEIN / G protein-coupled receptor / acetylcholine binding / seven transmembrane protein
Function / homology
Function and homology information


G-protein activation / Activation of the phototransduction cascade / Glucagon-type ligand receptors / Thromboxane signalling through TP receptor / Sensory perception of sweet, bitter, and umami (glutamate) taste / G beta:gamma signalling through PI3Kgamma / G beta:gamma signalling through CDC42 / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits ...G-protein activation / Activation of the phototransduction cascade / Glucagon-type ligand receptors / Thromboxane signalling through TP receptor / Sensory perception of sweet, bitter, and umami (glutamate) taste / G beta:gamma signalling through PI3Kgamma / G beta:gamma signalling through CDC42 / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Ca2+ pathway / G alpha (z) signalling events / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / Adrenaline,noradrenaline inhibits insulin secretion / ADP signalling through P2Y purinoceptor 12 / G alpha (q) signalling events / G alpha (i) signalling events / Thrombin signalling through proteinase activated receptors (PARs) / photoreceptor outer segment membrane / spectrin binding / alkylglycerophosphoethanolamine phosphodiesterase activity / photoreceptor outer segment / cardiac muscle cell apoptotic process / photoreceptor inner segment / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / 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 / ADP signalling through P2Y purinoceptor 12 / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / 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 / 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 / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / retina development in camera-type eye / positive regulation of cytosolic calcium ion concentration / cell body / GTPase binding / Ca2+ pathway / fibroblast proliferation / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / G alpha (i) signalling events / G alpha (s) signalling events / phospholipase C-activating G protein-coupled receptor signaling pathway / cellular response to hypoxia / G alpha (q) signalling events / Extra-nuclear estrogen signaling / cell population proliferation / G protein-coupled receptor signaling pathway / GTPase activity / synapse / dendrite / protein-containing complex binding / extracellular exosome / membrane / plasma membrane / cytoplasm
Similarity search - Function
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, 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 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
Similarity search - Domain/homology
: / ACETYLCHOLINE / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
Similarity search - Component
Biological speciesLama glama (llama)
Rattus norvegicus (Norway rat)
Homo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.06 Å
AuthorsBurger, W.A.C. / Mobbs, J.I. / Thal, D.M.
Funding support Australia, 2items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)1196951 Australia
Australian Research Council (ARC)LP180100560 Australia
CitationJournal: Nat Commun / Year: 2025
Title: Cryo-EM reveals an extrahelical allosteric binding site at the M mAChR.
Authors: Wessel A C Burger / Jesse I Mobbs / Bhavika Rana / Jinan Wang / Keya Joshi / Patrick R Gentry / Mahmuda Yeasmin / Hariprasad Venugopal / Aaron M Bender / Craig W Lindsley / Yinglong Miao / ...Authors: Wessel A C Burger / Jesse I Mobbs / Bhavika Rana / Jinan Wang / Keya Joshi / Patrick R Gentry / Mahmuda Yeasmin / Hariprasad Venugopal / Aaron M Bender / Craig W Lindsley / Yinglong Miao / Arthur Christopoulos / Celine Valant / David M Thal /
Abstract: The M muscarinic acetylcholine receptor (M mAChR) represents a promising therapeutic target for neurological disorders. However, the high conservation of its orthosteric binding site poses ...The M muscarinic acetylcholine receptor (M mAChR) represents a promising therapeutic target for neurological disorders. However, the high conservation of its orthosteric binding site poses significant challenges for drug development. While selective positive allosteric modulators (PAMs) offer a potential solution, a structural understanding of the M mAChR and its allosteric binding sites remains limited. Here, we present a 2.8 Å cryo-electron microscopy structure of the M mAChR complexed with heterotrimeric G protein and the agonist iperoxo, completing the active-state structural characterization of the mAChR family. To identify the binding site of M-selective PAMs, we implement an integrated approach combining mutagenesis, pharmacological assays, structural biology, and molecular dynamics simulations. Our mutagenesis studies reveal that selective M PAMs bind outside previously characterized M mAChR allosteric sites. Subsequently, we obtain a 2.1 Å structure of M mAChR co-bound with acetylcholine and the selective PAM VU6007678, revealing an allosteric pocket at the extrahelical interface between transmembrane domains 3 and 4 that is confirmed through mutagenesis and simulations. These findings demonstrate the diverse mechanisms of allosteric regulation in mAChRs and highlight the value of integrating pharmacological and structural approaches to identify allosteric binding sites.
History
DepositionNov 30, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 13, 2025Provider: repository / Type: Initial release
Revision 1.0Aug 13, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Aug 13, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Aug 13, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Aug 13, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Aug 13, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Aug 13, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Aug 13, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Oct 8, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
N: nanobody 35
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
G: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
A: Guanine nucleotide-binding protein Gq chimera
H: scFv16
R: Human M5 muscarinic acetylcholine receptor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)158,6698
Polymers158,0396
Non-polymers6312
Water1,38777
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Guanine nucleotide-binding protein ... , 3 types, 3 molecules BGA

#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 38534.062 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gnb1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P54311
#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 7563.750 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNG2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P59768
#4: Protein Guanine nucleotide-binding protein Gq chimera


Mass: 28039.793 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Trichoplusia ni (cabbage looper)

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Protein , 1 types, 1 molecules R

#6: Protein Human M5 muscarinic acetylcholine receptor


Mass: 39190.051 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CHRM5 / Production host: Spodoptera frugiperda (fall armyworm)

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Antibody , 2 types, 2 molecules NH

#1: Antibody nanobody 35


Mass: 16926.076 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#5: Antibody scFv16


Mass: 27784.896 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Trichoplusia ni (cabbage looper)

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Non-polymers , 3 types, 79 molecules

#7: Chemical ChemComp-A1BKS / VU6007678


Mass: 484.586 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C25H29FN4O3S / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-ACH / ACETYLCHOLINE


Mass: 146.207 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C7H16NO2
#9: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 77 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Human M5-miniGq-Gb1g2 complex / Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21rc1_5127 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 418794 / Symmetry type: POINT
RefinementHighest resolution: 2.06 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00510114
ELECTRON MICROSCOPYf_angle_d0.72913728
ELECTRON MICROSCOPYf_dihedral_angle_d13.0493637
ELECTRON MICROSCOPYf_chiral_restr0.0491553
ELECTRON MICROSCOPYf_plane_restr0.0071747

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