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- PDB-9uap: cryo-EM structure of ligand-free active-state M1 muscarinic acety... -

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Basic information

Entry
Database: PDB / ID: 9uap
Titlecryo-EM structure of ligand-free active-state M1 muscarinic acetylcholine receptor with alpha5 helix of G11 protein complex
Components
  • Guanine nucleotide-binding protein subunit alpha-11
  • M1 muscarinic acetylcholine receptor, de novo design protein
KeywordsMEMBRANE PROTEIN / GPCR / active-state / ligand-free / de novo protein
Function / homology
Function and homology information


regulation of melanocyte differentiation / phospholipase C-activating G protein-coupled acetylcholine receptor signaling pathway / endothelin receptor signaling pathway / Fatty Acids bound to GPR40 (FFAR1) regulate insulin secretion / Acetylcholine regulates insulin secretion / developmental pigmentation / phospholipase C-activating dopamine receptor signaling pathway / cellular response to pH / PLC beta mediated events / entrainment of circadian clock ...regulation of melanocyte differentiation / phospholipase C-activating G protein-coupled acetylcholine receptor signaling pathway / endothelin receptor signaling pathway / Fatty Acids bound to GPR40 (FFAR1) regulate insulin secretion / Acetylcholine regulates insulin secretion / developmental pigmentation / phospholipase C-activating dopamine receptor signaling pathway / cellular response to pH / PLC beta mediated events / entrainment of circadian clock / cranial skeletal system development / ligand-gated ion channel signaling pathway / phototransduction, visible light / action potential / photoreceptor outer segment / enzyme regulator activity / Turbulent (oscillatory, disturbed) flow shear stress activates signaling by PIEZO1 and integrins in endothelial cells / skeletal system development / G protein-coupled receptor binding / regulation of blood pressure / G-protein beta/gamma-subunit complex binding / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / positive regulation of insulin secretion / 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 / heart development / 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 / phospholipase C-activating G protein-coupled receptor signaling pathway / 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
Guanine nucleotide-binding protein subunit alpha-11
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.62 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 1, 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

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)45,2232
Polymers45,2232
Non-polymers00
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: 42070.730 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
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: Ligand-free active-state M1 muscarinic acetylcholine receptor with alpha5 helix of G11 protein complex/de novo design fusion protein
Type: COMPLEX / Entity ID: all / 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
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.20.1_4487model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.62 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 155176 / Symmetry type: POINT

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