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- EMDB-63988: cryo-EM structure of ligand-free active-state M1 muscarinic acety... -

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Basic information

Entry
Database: EMDB / ID: EMD-63988
Titlecryo-EM structure of ligand-free active-state M1 muscarinic acetylcholine receptor with alpha5 helix of G11 protein complex
Map data
Sample
  • Complex: Ligand-free active-state M1 muscarinic acetylcholine receptor with alpha5 helix of G11 protein complex/de novo design fusion protein
    • Protein or peptide: M1 muscarinic acetylcholine receptor, de novo design protein
    • Protein or peptide: Guanine nucleotide-binding protein subunit alpha-11
KeywordsGPCR / active-state / ligand-free / de novo protein / MEMBRANE PROTEIN
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 / cranial skeletal system development / endothelin receptor signaling pathway / 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 / cranial skeletal system development / endothelin receptor signaling pathway / 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
Guanine nucleotide-binding protein subunit alpha-11
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.62 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 1, 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_63988.png

Downloads & links

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Map

FileDownload / File: emd_63988.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 256 pix.
= 281.062 Å		1.1 Å/pix.
x 256 pix.
= 281.062 Å		1.1 Å/pix.
x 256 pix.
= 281.062 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0979 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-1.607326 - 3.0874395
Average (Standard dev.)-0.0003644062 (±0.046833407)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 281.0624 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_63988_msk_1.map
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Half map: #2

Fileemd_63988_half_map_1.map
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Half map: #1

Fileemd_63988_half_map_2.map
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Sample components

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Entire : Ligand-free active-state M1 muscarinic acetylcholine receptor wit...

EntireName: Ligand-free active-state M1 muscarinic acetylcholine receptor with alpha5 helix of G11 protein complex/de novo design fusion protein
Components
  • Complex: Ligand-free active-state M1 muscarinic acetylcholine receptor with alpha5 helix of G11 protein complex/de novo design fusion protein
    • Protein or peptide: M1 muscarinic acetylcholine receptor, de novo design protein
    • Protein or peptide: Guanine nucleotide-binding protein subunit alpha-11

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Supramolecule #1: Ligand-free active-state M1 muscarinic acetylcholine receptor wit...

SupramoleculeName: Ligand-free active-state M1 muscarinic acetylcholine receptor with alpha5 helix of G11 protein complex/de novo design fusion protein
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
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: 42.07073 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: VAFIGITTGL LSLATVTGNL LVLISFKVNT ELKTVNNYFL LSLACADLII GTFSMNLYTT YLLMGHWALG TLACDLWLAL DYVASNASV MNLLLISFDR YFSVTRPLSY RAKRTPRRAA LMIGLAWLVS FVLWAPAILF WQYLVGERTV LAGQCYIQFL S QPIITFGT ...String:
VAFIGITTGL LSLATVTGNL LVLISFKVNT ELKTVNNYFL LSLACADLII GTFSMNLYTT YLLMGHWALG TLACDLWLAL DYVASNASV MNLLLISFDR YFSVTRPLSY RAKRTPRRAA LMIGLAWLVS FVLWAPAILF WQYLVGERTV LAGQCYIQFL S QPIITFGT AMAAFYLPVT VMCTLYWRIY RETKRAGERL AKLLEKFEAL PLEDIVAALK ALLATNRPEI QLAVKTIVEN FP EIKKEAE KLTPEQKAKL AALEAQLADL PEELRKVLLS MYLTGLLYGG SEENRKRAIE KAARTLSAIL LAFILTWTPY NIM VLVSTF CKDCVPETLW ELGYWLCYVN STINPMCYAL CNKAFRDTFR LLLLCR

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Macromolecule #2: Guanine nucleotide-binding protein subunit alpha-11

MacromoleculeName: Guanine nucleotide-binding protein subunit alpha-11 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 3.15266 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
TPENIRFVFA AVKDTILQLN LKEYNLV

UniProtKB: Guanine nucleotide-binding protein subunit alpha-11

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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: 3.62 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 155176
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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