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- PDB-9uaz: Cryo-EM structure of the M1 muscarinic acetylcholine receptor bou... -

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Basic information

Entry
Database: PDB / ID: 9uaz
TitleCryo-EM structure of the M1 muscarinic acetylcholine receptor bound to atropine and nanobody NbA12
ComponentsM1 muscarinic acetylcholine receptor, de novo design protein
KeywordsMEMBRANE PROTEIN / GPCR / inactive-state / atropine / de novo protein / nanobody
Function / homology:
Function and homology information
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.29 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 2, 2025Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Oct 29, 2025Provider: repository / Type: Initial release
Revision 1.0Oct 29, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: M1 muscarinic acetylcholine receptor, de novo design protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)54,9812
Polymers54,6921
Non-polymers2891
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: 54691.621 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: Chemical ChemComp-A1EBT / [(1R,5S)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl] (2S)-3-oxidanyl-2-phenyl-propanoate / Atropine sulfate


Mass: 289.369 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H23NO3
Has ligand of interestY
Has protein modificationN

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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 the M1 muscarinic acetylcholine receptor bound to atropine and nanobody NbA12
Type: COMPLEX / Entity ID: #1 / 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
10cryoSPARCinitial Euler assignment
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.29 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 254687 / Symmetry type: POINT

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