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- EMDB-26591: CryoEM Structure of Inactive MOR Bound to Alvimopan and Mb6 -

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Basic information

Entry
Database: EMDB / ID: EMD-26591
TitleCryoEM Structure of Inactive MOR Bound to Alvimopan and Mb6
Map data
Sample
  • Complex: Complex of MOR and Nb6
    • Complex: MOR
      • Protein or peptide: Mu-type opioid receptor
    • Complex: Mb6
      • Protein or peptide: Megabody 6
  • Ligand: N-[(2S)-2-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethylpiperidin-1-yl]methyl}-3-phenylpropanoyl]glycine
  • Ligand: water
Function / homology
Function and homology information


Opioid Signalling / spine apparatus / beta-endorphin receptor activity / morphine receptor activity / negative regulation of Wnt protein secretion / negative regulation of adenylate cyclase-activating G protein-coupled receptor signaling pathway / Peptide ligand-binding receptors / adenylate cyclase-inhibiting opioid receptor signaling pathway / positive regulation of appetite / G-protein activation ...Opioid Signalling / spine apparatus / beta-endorphin receptor activity / morphine receptor activity / negative regulation of Wnt protein secretion / negative regulation of adenylate cyclase-activating G protein-coupled receptor signaling pathway / Peptide ligand-binding receptors / adenylate cyclase-inhibiting opioid receptor signaling pathway / positive regulation of appetite / G-protein activation / G protein-coupled opioid receptor activity / filamin binding / G protein-coupled opioid receptor signaling pathway / regulation of cellular response to stress / G alpha (i) signalling events / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / behavioral response to ethanol / negative regulation of nitric oxide biosynthetic process / neuropeptide binding / negative regulation of cAMP-mediated signaling / regulation of NMDA receptor activity / positive regulation of neurogenesis / eating behavior / negative regulation of cytosolic calcium ion concentration / transmission of nerve impulse / neuropeptide signaling pathway / G-protein alpha-subunit binding / GABA-ergic synapse / voltage-gated calcium channel activity / positive regulation of cAMP-mediated signaling / sensory perception of pain / dendrite membrane / excitatory postsynaptic potential / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / presynaptic modulation of chemical synaptic transmission / dendrite cytoplasm / locomotory behavior / G protein-coupled receptor activity / adenylate cyclase-activating dopamine receptor signaling pathway / G-protein beta-subunit binding / positive regulation of nitric oxide biosynthetic process / presynaptic membrane / phospholipase C-activating G protein-coupled receptor signaling pathway / positive regulation of cytosolic calcium ion concentration / perikaryon / postsynaptic membrane / response to ethanol / positive regulation of ERK1 and ERK2 cascade / endosome / neuron projection / G protein-coupled receptor signaling pathway / protein domain specific binding / axon / focal adhesion / dendrite / membrane / 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 speciesMus musculus (house mouse) / Lama glama (llama) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsRobertson MJ / Skiniotis G
Funding support United States, 1 items
OrganizationGrant numberCountry
The G. Harold and Leila Y. Mathers Foundation United States
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Structure determination of inactive-state GPCRs with a universal nanobody.
Authors: Michael J Robertson / Makaía M Papasergi-Scott / Feng He / Alpay B Seven / Justin G Meyerowitz / Ouliana Panova / Maria Claudia Peroto / Tao Che / Georgios Skiniotis /
Abstract: Cryogenic electron microscopy (cryo-EM) has widened the field of structure-based drug discovery by allowing for routine determination of membrane protein structures previously intractable. Despite ...Cryogenic electron microscopy (cryo-EM) has widened the field of structure-based drug discovery by allowing for routine determination of membrane protein structures previously intractable. Despite representing one of the largest classes of therapeutic targets, most inactive-state G protein-coupled receptors (GPCRs) have remained inaccessible for cryo-EM because their small size and membrane-embedded nature impedes projection alignment for high-resolution map reconstructions. Here we demonstrate that the same single-chain camelid antibody (nanobody) recognizing a grafted intracellular loop can be used to obtain cryo-EM structures of inactive-state GPCRs at resolutions comparable or better than those obtained by X-ray crystallography. Using this approach, we obtained structures of neurotensin 1 receptor bound to antagonist SR48692, μ-opioid receptor bound to alvimopan, apo somatostatin receptor 2 and histamine receptor 2 bound to famotidine. We expect this rapid, straightforward approach to facilitate the broad exploration of GPCR inactive states without the need for extensive engineering and crystallization.
History
DepositionApr 3, 2022-
Header (metadata) releaseJun 29, 2022-
Map releaseJun 29, 2022-
UpdateDec 28, 2022-
Current statusDec 28, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26591.png

Downloads & links

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Map

FileDownload / File: emd_26591.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.8677 Å
Density
Contour LevelBy AUTHOR: 0.7
Minimum - Maximum-3.332412 - 5.3181705
Average (Standard dev.)-0.0011871482 (±0.068818025)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 277.664 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Phenix model-free density modified map

Fileemd_26591_additional_1.map
AnnotationPhenix model-free density modified map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_26591_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_26591_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 : Complex of MOR and Nb6

EntireName: Complex of MOR and Nb6
Components
  • Complex: Complex of MOR and Nb6
    • Complex: MOR
      • Protein or peptide: Mu-type opioid receptor
    • Complex: Mb6
      • Protein or peptide: Megabody 6
  • Ligand: N-[(2S)-2-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethylpiperidin-1-yl]methyl}-3-phenylpropanoyl]glycine
  • Ligand: water

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Supramolecule #1: Complex of MOR and Nb6

SupramoleculeName: Complex of MOR and Nb6 / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #2: MOR

SupramoleculeName: MOR / type: complex / ID: 2 / Chimera: Yes / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #3: Mb6

SupramoleculeName: Mb6 / type: complex / ID: 3 / Chimera: Yes / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Lama glama (llama)

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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: Mus musculus (house mouse)
Molecular weightTheoretical: 47.920734 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MKTIIALSYI FCLVFADYKD DDDAMGPGNI SDCSDPLAPA SCSPAPGSWL NLSHVDGNQS DPCGPNRTGL GGSHSLCPQT GSPSMVTAI TIMALYSIVC VVGLFGNFLV MYVIVRYTKM KTATNIYIFN LALADALATS TLPFQSVNYL MGTWPFGNIL C KIVISIDY ...String:
MKTIIALSYI FCLVFADYKD DDDAMGPGNI SDCSDPLAPA SCSPAPGSWL NLSHVDGNQS DPCGPNRTGL GGSHSLCPQT GSPSMVTAI TIMALYSIVC VVGLFGNFLV MYVIVRYTKM KTATNIYIFN LALADALATS TLPFQSVNYL MGTWPFGNIL C KIVISIDY YNMFTSIFTL CTMSVDRYIA VCHPVKALDF RTPRNAKIVN VCNWILSSAI GLPVMFMATT KYRQGSIDCT LT FSHPTWY WENLLKICVF IFAFIMPVLI ITVCYGLMIL RLKSVRLLSG SREKDRNLRR ITRMVLVVVA VFIVCWTPIH IYV IIKALI TIPETTFQTV SWHFCIALGY TNSCLNPVLY AFLDENFKRC FREFCIPTSS TIEQQNSARI RQNTREHPST ANTV DRTNH QLENLEAETA PLPDIHHHHH H

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Macromolecule #2: Megabody 6

MacromoleculeName: Megabody 6 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 55.662078 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: QVQLQESGGG LVRKTTTSVI DTTNDAQNLL TQAQTIVNTL KDYCPILIAK SSSSNGGTNN ANTPSWQTAG GGKNSCATFG AEFSAASDM INNAQKIVQE TQQLSANQPK NITQPHNLNL NSPSSLTALA QKMLKNAQSQ AEILKLANQV ESDFNKLSSG H LKDYIGKC ...String:
QVQLQESGGG LVRKTTTSVI DTTNDAQNLL TQAQTIVNTL KDYCPILIAK SSSSNGGTNN ANTPSWQTAG GGKNSCATFG AEFSAASDM INNAQKIVQE TQQLSANQPK NITQPHNLNL NSPSSLTALA QKMLKNAQSQ AEILKLANQV ESDFNKLSSG H LKDYIGKC DASAISSANM TMQNQKNNWG NGCAGVEETQ SLLKTSAADF NNQTPQINQA QNLANTLIQE LGNNTYEQLS RL LTNDNGT NSKTSAQAIN QAVNNLNERA KTLAGGTTNS PAYQATLLAL RSVLGLWNSM GYAVICGGYT KSPGENNQKD FHY TDENGN GTTINCGGST NSNGTHSYNG TNTLKADKNV SLSIEQYEKI HEAYQILSKA LKQAGLAPLN SKGEKLEAHV TTSK PSLRL SCAASGTIFR LYDMGWYRRV SGNQRELVAS ITSGGSTKYG DSVKGRFTIS RDNAKNTVYL QMSSLKPEDT AVYYC NAEY RTGIWEELLD GWGQGTQVTV SSHHHHHHEP EA

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Macromolecule #3: N-[(2S)-2-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethylpiperidin-1-y...

MacromoleculeName: N-[(2S)-2-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethylpiperidin-1-yl]methyl}-3-phenylpropanoyl]glycine
type: ligand / ID: 3 / Number of copies: 1 / Formula: NG0
Molecular weightTheoretical: 424.533 Da
Chemical component information

ChemComp-NG0:
N-[(2S)-2-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethylpiperidin-1-yl]methyl}-3-phenylpropanoyl]glycine / antagonist*YM / Alvimopan

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 4 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.7000000000000001 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 61.38 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: OTHER
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 301332

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