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- EMDB-48525: Inactive Mu-Opioid Receptor with Nb6M, NabFab, and isoquinuclidin... -

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Basic information

Entry
Database: EMDB / ID: EMD-48525
TitleInactive Mu-Opioid Receptor with Nb6M, NabFab, and isoquinuclidine compound#020_E1
Map data
Sample
  • Complex: Human Mu-Opioid Receptor
    • Protein or peptide: Mu-type opioid receptor
    • Protein or peptide: Nanobody 6M
    • Protein or peptide: NabFab Heavy Chain
    • Protein or peptide: NabFab Light Chain
  • Ligand: methyl (1S,3R,4S,6S,8M)-2-[(1-ethyl-1H-pyrazol-4-yl)methyl]-8-(3-hydroxyphenyl)-3,4-dimethyl-2-azabicyclo[2.2.2]oct-7-ene-6-carboxylate
KeywordsG-protein coupled receptor / Mu-opioid / isoquinuclidine / antagonist / nanobodies / fabs / MEMBRANE PROTEIN
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 / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / negative regulation of nitric oxide biosynthetic process / 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 / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / negative regulation of nitric oxide biosynthetic process / sensory perception / neuropeptide binding / regulation of NMDA receptor activity / 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 / 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 / perikaryon / G alpha (i) signalling events / phospholipase C-activating G protein-coupled receptor signaling pathway / positive regulation of ERK1 and ERK2 cascade / neuron projection / endosome / G protein-coupled receptor signaling pathway / axon / negative regulation of cell population proliferation / dendrite / synapse / endoplasmic reticulum / Golgi apparatus / plasma membrane
Similarity search - Function
Mu opioid receptor / Opioid receptor / 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
Mu-type opioid receptor
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsKim JY / Vigneron SF / Billesbolle C / Manglik A / Shoichet BK
Funding support United States, 3 items
OrganizationGrant numberCountry
Defense Advanced Research Projects Agency (DARPA)HR0011-19-2-0020 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM122481 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM122473 United States
CitationJournal: bioRxiv / Year: 2025
Title: Docking 14 million virtual isoquinuclidines against the mu and kappa opioid receptors reveals dual antagonists-inverse agonists with reduced withdrawal effects.
Authors: Seth F Vigneron / Shohei Ohno / Joao Braz / Joseph Y Kim / Oh Sang Kweon / Chase Webb / Christian Billesbølle / Karnika Bhardwaj / John Irwin / Aashish Manglik / Allan I Basbaum / Jonathan ...Authors: Seth F Vigneron / Shohei Ohno / Joao Braz / Joseph Y Kim / Oh Sang Kweon / Chase Webb / Christian Billesbølle / Karnika Bhardwaj / John Irwin / Aashish Manglik / Allan I Basbaum / Jonathan A Ellman / Brian K Shoichet /
Abstract: Large library docking of tangible molecules has revealed potent ligands across many targets. While make-on-demand libraries now exceed 75 billion enumerated molecules, their synthetic routes are ...Large library docking of tangible molecules has revealed potent ligands across many targets. While make-on-demand libraries now exceed 75 billion enumerated molecules, their synthetic routes are dominated by a few reaction types, reducing diversity and inevitably leaving many interesting bioactive-like chemotypes unexplored. Here, we investigate the large-scale enumeration and targeted docking of isoquinuclidines. These "natural-product-like" molecules are rare in the current libraries and are functionally congested, making them interesting as receptor probes. Using a modular, four-component reaction scheme, we built and docked a virtual library of over 14.6 million isoquinuclidines against both the μ- and -opioid receptors (MOR and KOR, respectively). Synthesis and experimental testing of 18 prioritized compounds found nine ligands with low μM affinities. Structure-based optimization revealed low- and sub-nM antagonists and inverse agonists targeting both receptors. Cryo-electron microscopy (cryoEM) structures illuminate the origins of activity on each target. In mouse behavioral studies, a potent member of the series with joint MOR-antagonist and KOR-inverse-agonist activity reversed morphine-induced analgesia, phenocopying the MOR-selective anti-overdose agent naloxone. Encouragingly, the new molecule induced less severe opioid-induced withdrawal symptoms compared to naloxone during withdrawal precipitation, and did not induce conditioned-place aversion, likely reflecting a reduction of dysphoria due to the compound's KOR-inverse agonism. The strengths and weaknesses of bespoke library docking, and of docking for opioid receptor polypharmacology, will be considered.
History
DepositionJan 3, 2025-
Header (metadata) releaseFeb 12, 2025-
Map releaseFeb 12, 2025-
UpdateFeb 12, 2025-
Current statusFeb 12, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_48525.png

Downloads & links

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Map

FileDownload / File: emd_48525.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 360 pix.
= 294.804 Å		0.82 Å/pix.
x 360 pix.
= 294.804 Å		0.82 Å/pix.
x 360 pix.
= 294.804 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8189 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.371
Minimum - Maximum-2.58749 - 3.3918226
Average (Standard dev.)0.000026449763 (±0.03809909)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 294.804 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_48525_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_48525_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Human Mu-Opioid Receptor

EntireName: Human Mu-Opioid Receptor
Components
  • Complex: Human Mu-Opioid Receptor
    • Protein or peptide: Mu-type opioid receptor
    • Protein or peptide: Nanobody 6M
    • Protein or peptide: NabFab Heavy Chain
    • Protein or peptide: NabFab Light Chain
  • Ligand: methyl (1S,3R,4S,6S,8M)-2-[(1-ethyl-1H-pyrazol-4-yl)methyl]-8-(3-hydroxyphenyl)-3,4-dimethyl-2-azabicyclo[2.2.2]oct-7-ene-6-carboxylate

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Supramolecule #1: Human Mu-Opioid Receptor

SupramoleculeName: Human Mu-Opioid Receptor / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 46 MDa

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Macromolecule #1: Mu-type opioid receptor

MacromoleculeName: Mu-type opioid receptor / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 46.081359 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DYKDDDDASI DMDSSAAPTN ASNCTDALAY SSCSPAPSPG SWVNLSHLDG NLSDPCGPNR TDLGGRDSLC PPTGSPSMIT AITIMALYS IVCVVGLFGN FLVMYVIVRY TKMKTATNIY IFNLALADAL ATSTLPFQSV NYLMGTWPFG TILCKIVISI D YYNMFTSI ...String:
DYKDDDDASI DMDSSAAPTN ASNCTDALAY SSCSPAPSPG SWVNLSHLDG NLSDPCGPNR TDLGGRDSLC PPTGSPSMIT AITIMALYS IVCVVGLFGN FLVMYVIVRY TKMKTATNIY IFNLALADAL ATSTLPFQSV NYLMGTWPFG TILCKIVISI D YYNMFTSI FTLCTMSVDR YIAVCHPVKA LDFRTPRNAK IINVCNWILS SAIGLPVMFM ATTKYRQGSI DCTLTFSHPT WY WENLLKI CVFIFAFIMP VLIITVCYGL MILRLKSVRL LSGSREKDRN LRRITRMVLV VVAVFIVCWT PIHIYVIIKA LVT IPETTF QTVSWHFCIA LGYTNSCLNP VLYAFLDENF KRCFREFCIP TSSNIEQQNS TRIRQNTRDH PSTANTVDRT NHQL ENLEA ETAPLP

UniProtKB: Mu-type opioid receptor

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Macromolecule #2: Nanobody 6M

MacromoleculeName: Nanobody 6M / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 14.418919 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QRQLVESGGG LVQPGGSLRL SCAASGTIFR LYDMGWFRQA PGKEREGVAS ITSGGSTKYA DSVKGRFTIS RDNAKNTVYL QMNSLEPED TAVYYCNAEY RTGIWEELLD GWGKGTPVTV SSHHHHHHEP EA

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Macromolecule #3: NabFab Heavy Chain

MacromoleculeName: NabFab Heavy Chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 25.684463 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NFSYYSIHWV RQAPGKGLEW VAYISSSSSY TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARGYQYWQYH ASWYWNGGLD YWGQGTLVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL V KDYFPEPV ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NFSYYSIHWV RQAPGKGLEW VAYISSSSSY TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARGYQYWQYH ASWYWNGGLD YWGQGTLVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL V KDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCDKTH T

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Macromolecule #4: NabFab Light Chain

MacromoleculeName: NabFab Light Chain / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 23.258783 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSSSSLITF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSSSSLITF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

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Macromolecule #5: methyl (1S,3R,4S,6S,8M)-2-[(1-ethyl-1H-pyrazol-4-yl)methyl]-8-(3-...

MacromoleculeName: methyl (1S,3R,4S,6S,8M)-2-[(1-ethyl-1H-pyrazol-4-yl)methyl]-8-(3-hydroxyphenyl)-3,4-dimethyl-2-azabicyclo[2.2.2]oct-7-ene-6-carboxylate
type: ligand / ID: 5 / Number of copies: 1 / Formula: A1BNM
Molecular weightTheoretical: 395.495 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
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsPhase plate: OTHER
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 47.7 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 259816
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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