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- EMDB-64050: cryo-EM structure of M1 muscarinic acetylcholine receptor-alpha5 ... -

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Basic information

Entry
Database: EMDB / ID: EMD-64050
Titlecryo-EM structure of M1 muscarinic acetylcholine receptor-alpha5 helix of G11 protein complex bound to iperoxo and nanobody Nb1B4
Map data
Sample
  • Complex: 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
    • Protein or peptide: M1 muscarinic acetylcholine receptor, de novo design protein
    • Protein or peptide: Guanine nucleotide-binding protein subunit alpha-11
    • Protein or peptide: Nanobody Nb1B4
  • Ligand: 4-(4,5-dihydro-1,2-oxazol-3-yloxy)-N,N,N-trimethylbut-2-yn-1-aminium
KeywordsGPCR / active-state / nanobody / de novo protein / MEMBRANE PROTEIN/IMMUNE SYSTEM / MEMBRANE PROTEIN-IMMUNE SYSTEM complex
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) / Vicugna pacos (alpaca)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.88 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 5, 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_64050.png

Downloads & links

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Map

FileDownload / File: emd_64050.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.)
1.08 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
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-4.1961503 - 5.653426
Average (Standard dev.)0.00056715833 (±0.07878575)
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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Mask #1

Fileemd_64050_msk_1.map
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Half map: #2

Fileemd_64050_half_map_1.map
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Half map: #1

Fileemd_64050_half_map_2.map
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Sample components

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Entire : cryo-EM structure of M1 muscarinic acetylcholine receptor-alpha5 ...

EntireName: 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
Components
  • Complex: 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
    • Protein or peptide: M1 muscarinic acetylcholine receptor, de novo design protein
    • Protein or peptide: Guanine nucleotide-binding protein subunit alpha-11
    • Protein or peptide: Nanobody Nb1B4
  • Ligand: 4-(4,5-dihydro-1,2-oxazol-3-yloxy)-N,N,N-trimethylbut-2-yn-1-aminium

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Supramolecule #1: cryo-EM structure of M1 muscarinic acetylcholine receptor-alpha5 ...

SupramoleculeName: 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 / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
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: 49.317816 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 RIYRETKRAG ERLAKLLEKF EA LPLEDIV AALKALLATN RPEIQLAVKT IVENFPEIKK EAEKLTPEQK AKLAALEAQL ADLPEELRKV LLSMYLTGLL YGG SEENRK RAIEKAARTL SAILLAFILT WTPYNIMVLV STFCKDCVPE TLWELGYWLC YVNSTINPMC YALCNKAFRD TFRL LLLCR WDKRRWRKIP KRPGSVHRTP SRQCHHHHHH

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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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Macromolecule #3: Nanobody Nb1B4

MacromoleculeName: Nanobody Nb1B4 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Molecular weightTheoretical: 13.766307 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
QVQLQESGGG LVQAGGSLRL SCAASGSIFY GYYMGWYRQA PGKEREFVAT INYGASTNYA DSVKGRFTIS RDNAKNTVYL QMNSLKPED TAVYYCAVRR VYVYVQRYYH YYWGQGTQVT VSS

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Macromolecule #4: 4-(4,5-dihydro-1,2-oxazol-3-yloxy)-N,N,N-trimethylbut-2-yn-1-aminium

MacromoleculeName: 4-(4,5-dihydro-1,2-oxazol-3-yloxy)-N,N,N-trimethylbut-2-yn-1-aminium
type: ligand / ID: 4 / Number of copies: 1 / Formula: IXO
Molecular weightTheoretical: 197.254 Da
Chemical component information

ChemComp-IXO:
4-(4,5-dihydro-1,2-oxazol-3-yloxy)-N,N,N-trimethylbut-2-yn-1-aminium

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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: 2.88 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 191008
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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