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- EMDB-63998: Cryo-EM structure of the M1 muscarinic acetylcholine receptor bou... -

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Entry
Database: EMDB / ID: EMD-63998
TitleCryo-EM structure of the M1 muscarinic acetylcholine receptor bound to atropine and nanobody NbA12
Map data
Sample
  • Complex: Cryo-EM structure of the M1 muscarinic acetylcholine receptor bound to atropine and nanobody NbA12
    • Protein or peptide: M1 muscarinic acetylcholine receptor, de novo design protein
  • Ligand: [(1R,5S)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl] (2S)-3-oxidanyl-2-phenyl-propanoate
KeywordsGPCR / inactive-state / atropine / de novo protein / MEMBRANE PROTEIN / nanobody
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.29 Å
AuthorsZhang X / Gao K / Liu X
Funding support China, 1 items
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, 2025-
Header (metadata) releaseOct 29, 2025-
Map releaseOct 29, 2025-
UpdateMay 13, 2026-
Current statusMay 13, 2026Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_63998.png

Downloads & links

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Map

FileDownload / File: emd_63998.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.)
1.08 Å/pix.
x 256 pix.
= 277.12 Å		1.08 Å/pix.
x 256 pix.
= 277.12 Å		1.08 Å/pix.
x 256 pix.
= 277.12 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0825 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-3.7509346 - 4.873107
Average (Standard dev.)-0.0014058581 (±0.059342522)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 277.12 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_63998_half_map_1.map
Projections & Slices
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Slices (1/2)
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Half map: #2

Fileemd_63998_half_map_2.map
Projections & Slices
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Sample components

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Entire : Cryo-EM structure of the M1 muscarinic acetylcholine receptor bou...

EntireName: Cryo-EM structure of the M1 muscarinic acetylcholine receptor bound to atropine and nanobody NbA12
Components
  • Complex: Cryo-EM structure of the M1 muscarinic acetylcholine receptor bound to atropine and nanobody NbA12
    • Protein or peptide: M1 muscarinic acetylcholine receptor, de novo design protein
  • Ligand: [(1R,5S)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl] (2S)-3-oxidanyl-2-phenyl-propanoate

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Supramolecule #1: Cryo-EM structure of the M1 muscarinic acetylcholine receptor bou...

SupramoleculeName: Cryo-EM structure of the M1 muscarinic acetylcholine receptor bound to atropine and nanobody NbA12
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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

MacromoleculeName: M1 muscarinic acetylcholine receptor, de novo design protein
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 54.691621 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DYKDDDDAAA QTSAPPAVSP QITVLAPGKG PWQVAFIGIT TGLLSLATVT GNLLVLISFK VNTELKTVNN YFLLSLACAD LIIGTFSMN LYTTYLLMGH WALGTLACDL WLALDYVASN ASVMNLLLIS FDRYFSVTRP LSYRAKRTPR RAALMIGLAW L VSFVLWAP ...String:
DYKDDDDAAA QTSAPPAVSP QITVLAPGKG PWQVAFIGIT TGLLSLATVT GNLLVLISFK VNTELKTVNN YFLLSLACAD LIIGTFSMN LYTTYLLMGH WALGTLACDL WLALDYVASN ASVMNLLLIS FDRYFSVTRP LSYRAKRTPR RAALMIGLAW L VSFVLWAP AILFWQYLVG ERTVLAGQCY IQFLSQPIIT FGTAMAAFYL PVTVMCTLYW RIYRFRRRGA EALERAFSLE DD KEALLAA LDALAEAFAD DAELTALLAL LRRLLEDPDL PADELAALRA ALTRFPEFRE ALLALLDRYL ATRDLADARD LVW ALVLAI ASDPRYRPAV AAMIAFGDAE VLRAGLLRGA EALGLPGGEA LVEEIMAEAE KEKKAARTLS AILLAFILTW TPYN IMVLV STFCKDCVPE TLWELGYWLC YVNSTINPMC YALCNKAFRD TFRLLLLCRW DKRRWRKIPK RPGSVHRTPS RQCHH HHHH HHH

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Macromolecule #2: [(1R,5S)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl] (2S)-3-oxidanyl-...

MacromoleculeName: [(1R,5S)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl] (2S)-3-oxidanyl-2-phenyl-propanoate
type: ligand / ID: 2 / Number of copies: 1 / Formula: A1EBT
Molecular weightTheoretical: 289.369 Da

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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 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.1 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING ONLY
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.29 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 254687
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD

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