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- PDB-9ucp: cryo-EM structure of M1 muscarinic acetylcholine receptor-alpha5 ... -

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Basic information

Entry
Database: PDB / ID: 9ucp
Titlecryo-EM structure of M1 muscarinic acetylcholine receptor-alpha5 helix of G11 protein complex bound to iperoxo and nanobody Nb1B4
Components
  • Guanine nucleotide-binding protein subunit alpha-11
  • M1 muscarinic acetylcholine receptor, de novo design protein
  • Nanobody Nb1B4
KeywordsMEMBRANE PROTEIN/IMMUNE SYSTEM / GPCR / active-state / nanobody / de novo protein / MEMBRANE PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


phospholipase C-activating G protein-coupled acetylcholine receptor signaling pathway / Fatty Acids bound to GPR40 (FFAR1) regulate insulin secretion / phospholipase C-activating dopamine receptor signaling pathway / Acetylcholine regulates insulin secretion / endothelin receptor signaling pathway / cranial skeletal system development / PLC beta mediated events / entrainment of circadian clock / phototransduction, visible light / action potential ...phospholipase C-activating G protein-coupled acetylcholine receptor signaling pathway / Fatty Acids bound to GPR40 (FFAR1) regulate insulin secretion / phospholipase C-activating dopamine receptor signaling pathway / Acetylcholine regulates insulin secretion / endothelin receptor signaling pathway / cranial skeletal system development / PLC beta mediated events / entrainment of circadian clock / phototransduction, visible light / action potential / photoreceptor outer segment / Turbulent (oscillatory, disturbed) flow shear stress activates signaling by PIEZO1 and integrins in endothelial cells / regulation of blood pressure / G protein-coupled receptor binding / G-protein beta/gamma-subunit complex binding / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / phospholipase C-activating G protein-coupled receptor signaling pathway / G protein-coupled acetylcholine receptor signaling pathway / Thromboxane signalling through TP receptor / G-protein activation / ADP signalling through P2Y purinoceptor 1 / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / heterotrimeric G-protein complex / Thrombin signalling through proteinase activated receptors (PARs) / G protein activity / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / G alpha (q) signalling events / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / lysosomal membrane / GTPase activity / synapse / GTP binding / signal transduction / extracellular exosome / metal ion binding / plasma membrane / cytoplasm
Similarity search - Function
G-protein alpha subunit, group Q / G protein alpha subunit, helical insertion / G protein alpha subunit / Guanine nucleotide binding protein (G-protein), alpha subunit / G-protein alpha subunit / G-alpha domain profile. / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Chem-IXO / Guanine nucleotide-binding protein subunit alpha-11
Similarity search - Component
Biological speciesHomo sapiens (human)
Vicugna pacos (alpaca)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.88 Å
AuthorsZhang, X. / Gao, K. / Liu, X.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32122041 China
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Extracellular nanobody screening using conformationally stable GPCR variants.
Authors: Xin Zhang / Kaixuan Gao / Jia Nie / Hengyu Meng / Xiaoou Sun / Jiawei Zhao / Xiangyu Liu /
Abstract: G protein-coupled receptors (GPCRs) are prominent drug targets that have attracted intensive efforts in drug screening. Binding-based screening methods for GPCR ligands often require conformationally ...G protein-coupled receptors (GPCRs) are prominent drug targets that have attracted intensive efforts in drug screening. Binding-based screening methods for GPCR ligands often require conformationally stable, purified receptors. However, obtaining large quantities of GPCRs in stable states, particularly with unoccupied extracellular ligand-binding pockets and especially in their active conformations, remains challenging due to the inherent dynamic nature of these receptors. To address this challenge, we propose a universal approach for stabilizing GPCRs in specific conformations. Using the M1 muscarinic acetylcholine receptor (M1R) as a model, we successfully stabilized M1R in its active conformation through de novo design of a fusion protein, and further demonstrated the generalizability of this strategy by applying it to other GPCRs. We screened a synthetic yeast display library of nanobodies against both the stabilized active-state and previously reported inactive-state M1R, identifying several nanobodies that specifically recognize each conformation. This method not only facilitates the stabilization of GPCRs in desired states but also provides valuable tools for developing more selective therapeutic agents, enhancing drug discovery efficiency and specificity.
History
DepositionApr 5, 2025Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Oct 29, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: M1 muscarinic acetylcholine receptor, de novo design protein
B: Guanine nucleotide-binding protein subunit alpha-11
C: Nanobody Nb1B4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)66,4344
Polymers66,2373
Non-polymers1971
Water00
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

#1: Protein M1 muscarinic acetylcholine receptor, de novo design protein


Mass: 49317.816 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Spodoptera frugiperda (fall armyworm)
#2: Protein/peptide Guanine nucleotide-binding protein subunit alpha-11 / G alpha-11 / G-protein subunit alpha-11 / Guanine nucleotide-binding protein G(y) subunit alpha


Mass: 3152.660 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNA11, GA11 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P29992, Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement
#3: Antibody Nanobody Nb1B4


Mass: 13766.307 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Production host: Homo sapiens (human)
#4: Chemical ChemComp-IXO / 4-(4,5-dihydro-1,2-oxazol-3-yloxy)-N,N,N-trimethylbut-2-yn-1-aminium / Iperoxo


Mass: 197.254 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N2O2 / Feature type: SUBJECT OF INVESTIGATION
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: cryo-EM structure of M1 muscarinic acetylcholine receptor-alpha5 helix of G11 protein complex bound to iperoxo and nanobody Nb1B4/de novo design fusion protein
Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 1100 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 2.88 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 191008 / Symmetry type: POINT

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