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- PDB-9pxu: Inactive-state naloxone-mu opioid receptor nanobody6 complex -

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Basic information

Entry
Database: PDB / ID: 9pxu
TitleInactive-state naloxone-mu opioid receptor nanobody6 complex
Components
  • Anti-fab nanobody
  • Nab-fab heavy chain
  • Nab-fab light chain
  • Nanobody6
  • Soluble cytochrome b562,Mu-type opioid receptor
KeywordsMEMBRANE PROTEIN / G protein coupled receptor / Mu Opioid receptor / Naloxone
Function / homology
Function and homology information


Opioid Signalling / beta-endorphin receptor activity / morphine receptor activity / negative regulation of Wnt protein secretion / regulation of cellular response to stress / G protein-coupled opioid receptor signaling pathway / behavioral response to ethanol / negative regulation of nitric oxide biosynthetic process / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / sensory perception ...Opioid Signalling / beta-endorphin receptor activity / morphine receptor activity / negative regulation of Wnt protein secretion / regulation of cellular response to stress / G protein-coupled opioid receptor signaling pathway / behavioral response to ethanol / negative regulation of nitric oxide biosynthetic process / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / sensory perception / regulation of NMDA receptor activity / neuropeptide binding / positive regulation of neurogenesis / negative regulation of cytosolic calcium ion concentration / G-protein alpha-subunit binding / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / neuropeptide signaling pathway / voltage-gated calcium channel activity / MECP2 regulates neuronal receptors and channels / sensory perception of pain / Peptide ligand-binding receptors / electron transport chain / G protein-coupled receptor activity / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / G-protein activation / G-protein beta-subunit binding / Interleukin-4 and Interleukin-13 signaling / G alpha (i) signalling events / perikaryon / phospholipase C-activating G protein-coupled receptor signaling pathway / electron transfer activity / periplasmic space / positive regulation of ERK1 and ERK2 cascade / endosome / neuron projection / iron ion binding / G protein-coupled receptor signaling pathway / axon / negative regulation of cell population proliferation / heme binding / synapse / dendrite / endoplasmic reticulum / Golgi apparatus / plasma membrane
Similarity search - Function
Mu opioid receptor / Opioid receptor / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / G-protein coupled receptors family 1 signature. / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family)
Similarity search - Domain/homology
: / Soluble cytochrome b562 / Mu-type opioid receptor
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsGati, C. / Khan, S. / Han, G.W.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM144965 United States
National Institutes of Health/National Center for Complementary and Integrative Health (NIH/NCCIH)AT012075 United States
CitationJournal: Nature / Year: 2025
Title: Structural snapshots capture nucleotide release at the μ-opioid receptor.
Authors: Saif Khan / Aaliyah S Tyson / Mohsen Ranjbar / Zixin Zhang / Jaskaran Singh / Gye Won Han / Cornelius Gati /
Abstract: As a member of the G protein-coupled receptor superfamily, the μ-opioid receptor (MOR) activates heterotrimeric G proteins by opening the Gα α-helical domain (AHD) to enable GDP-GTP exchange, ...As a member of the G protein-coupled receptor superfamily, the μ-opioid receptor (MOR) activates heterotrimeric G proteins by opening the Gα α-helical domain (AHD) to enable GDP-GTP exchange, with GDP release representing the rate-limiting step. Here, using pharmacological assays, we show that agonist efficacy correlates with decreased GDP affinity, promoting GTP exchange, whereas antagonists increase GDP affinity, dampening activation. Further investigating this phenomenon, we provide 8 unique structural models and 16 cryogenic electron microscopy maps of MOR with naloxone or loperamide, capturing several intermediate conformations along the activation pathway. These include four GDP-bound states with previously undescribed receptor-G protein interfaces, AHD arrangements and transitions in the nucleotide-binding pocket required for GDP release. Naloxone stalls MOR in a 'latent' state, whereas loperamide promotes an 'engaged' state, which is structurally poised for opening of the AHD domain and subsequent GDP release. These findings, supported by molecular dynamics simulations, identify GDP-bound intermediates and AHD conformations as key determinants of nucleotide exchange rates, providing structural and mechanistic insights into G protein activation and ligand efficacy with broad implications for G protein-coupled receptor pharmacology.
History
DepositionAug 6, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 12, 2025Provider: repository / Type: Initial release
Revision 1.1Nov 19, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
R: Soluble cytochrome b562,Mu-type opioid receptor
C: Nanobody6
K: Anti-fab nanobody
L: Nab-fab light chain
H: Nab-fab heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)147,0277
Polymers146,6775
Non-polymers3502
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

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Protein , 1 types, 1 molecules R

#1: Protein Soluble cytochrome b562,Mu-type opioid receptor / Cytochrome b-562 / M-OR-1 / MOR-1 / Mu opiate receptor / Mu opioid receptor / MOP / hMOP


Mass: 69550.344 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: cybC, OPRM1, MOR1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P0ABE7, UniProt: P35372

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Antibody , 4 types, 4 molecules CKLH

#2: Antibody Nanobody6


Mass: 14418.919 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#3: Antibody Anti-fab nanobody


Mass: 13764.110 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#4: Antibody Nab-fab light chain


Mass: 23258.783 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#5: Antibody Nab-fab heavy chain


Mass: 25684.463 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)

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Non-polymers , 2 types, 2 molecules

#6: Chemical ChemComp-A1APV / Naloxone / 3,14-dihydroxy-17-(prop-2-en-1-yl)-5alpha-4,5-epoxymorphinan-6-one


Mass: 327.374 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H21NO4 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na

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Details

Has ligand of interestY
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: Naloxone-mu opioid receptor in complex with Nb6M, NabFab and AntiFab Nb
Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightValue: 130 kDa/nm / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
140 mMHEPESHEPES1
2100 mMsodium chlorideNaCl1
30.00075 %detergentLMNG1
40.00025 %detergentGDN1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 K

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 51 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2EPU2image acquisition
4cryoSPARCv4.3.1CTF correction
10cryoSPARCv4.3.1initial Euler assignment
11cryoSPARCv4.3.1final Euler assignment
12cryoSPARCv4.3.1classification
13cryoSPARCv4.3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 180003 / Symmetry type: POINT
RefinementHighest resolution: 3.4 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037345
ELECTRON MICROSCOPYf_angle_d0.73310043
ELECTRON MICROSCOPYf_dihedral_angle_d4.6931055
ELECTRON MICROSCOPYf_chiral_restr0.0481157
ELECTRON MICROSCOPYf_plane_restr0.0061261

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