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- EMDB-20487: Human alpha3beta4 nicotinic acetylcholine receptor in complex wit... -

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Basic information

Entry
Database: EMDB / ID: EMD-20487
TitleHuman alpha3beta4 nicotinic acetylcholine receptor in complex with nicotine
Map data
Sample
  • Complex: Human alpha3beta4 nicotinic receptor in complex with Fab fragments and bound to nicotine
    • Complex: Human alpha3beta4 nicotinic acetylcholine receptor
      • Complex: Neuronal acetylcholine receptor subunit alpha-3
        • Protein or peptide: Fusion protein of Neuronal acetylcholine receptor subunit alpha-3 and Soluble cytochrome b562
      • Complex: Neuronal acetylcholine receptor subunit beta-4
        • Protein or peptide: Fusion protein of Neuronal acetylcholine receptor subunit beta-4 and Soluble cytochrome b562
    • Complex: IgG2b/kappa antibody
      • Complex: IgG2b heavy chain
        • Protein or peptide: IgG2b Fab heavy chain
      • Complex: Kappa Fab light chain
        • Protein or peptide: Kappa Fab light chain
  • Ligand: (S)-3-(1-METHYLPYRROLIDIN-2-YL)PYRIDINE
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: SODIUM IONSodium
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: nonane
  • Ligand: water
Function / homology
Function and homology information


regulation of acetylcholine secretion, neurotransmission / synaptic transmission involved in micturition / Highly sodium permeable postsynaptic acetylcholine nicotinic receptors / positive regulation of transmission of nerve impulse / Highly calcium permeable nicotinic acetylcholine receptors / acetylcholine receptor activity / response to acetylcholine / Highly calcium permeable postsynaptic nicotinic acetylcholine receptors / excitatory extracellular ligand-gated monoatomic ion channel activity / acetylcholine-gated channel complex ...regulation of acetylcholine secretion, neurotransmission / synaptic transmission involved in micturition / Highly sodium permeable postsynaptic acetylcholine nicotinic receptors / positive regulation of transmission of nerve impulse / Highly calcium permeable nicotinic acetylcholine receptors / acetylcholine receptor activity / response to acetylcholine / Highly calcium permeable postsynaptic nicotinic acetylcholine receptors / excitatory extracellular ligand-gated monoatomic ion channel activity / acetylcholine-gated channel complex / regulation of smooth muscle contraction / cholinergic synapse / regulation of neurotransmitter secretion / behavioral response to nicotine / acetylcholine-gated monoatomic cation-selective channel activity / synaptic transmission, cholinergic / neurotransmitter receptor activity / nervous system process / acetylcholine binding / acetylcholine receptor signaling pathway / activation of transmembrane receptor protein tyrosine kinase activity / regulation of dendrite morphogenesis / plasma membrane raft / membrane depolarization / tertiary granule membrane / ligand-gated monoatomic ion channel activity / neuronal action potential / smooth muscle contraction / specific granule membrane / monoatomic ion transport / excitatory postsynaptic potential / regulation of membrane potential / monoatomic ion transmembrane transport / locomotory behavior / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / response to nicotine / nervous system development / chemical synaptic transmission / postsynaptic membrane / postsynaptic density / electron transfer activity / periplasmic space / neuron projection / nuclear speck / iron ion binding / dendrite / neuronal cell body / synapse / heme binding / Neutrophil degranulation / Golgi apparatus / endoplasmic reticulum / signal transduction / membrane / plasma membrane
Similarity search - Function
Nicotinic acetylcholine receptor / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channels signature. / Neurotransmitter-gated ion-channel transmembrane domain / Neurotransmitter-gated ion-channel transmembrane region / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Neurotransmitter-gated ion-channel transmembrane domain superfamily / Neuronal acetylcholine receptor ...Nicotinic acetylcholine receptor / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channels signature. / Neurotransmitter-gated ion-channel transmembrane domain / Neurotransmitter-gated ion-channel transmembrane region / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Neurotransmitter-gated ion-channel transmembrane domain superfamily / Neuronal acetylcholine receptor / Neurotransmitter-gated ion-channel / Neurotransmitter-gated ion-channel ligand-binding domain / Neurotransmitter-gated ion-channel ligand-binding domain superfamily / Neurotransmitter-gated ion-channel ligand binding domain
Similarity search - Domain/homology
Soluble cytochrome b562 / Neuronal acetylcholine receptor subunit beta-4 / Neuronal acetylcholine receptor subunit alpha-3
Similarity search - Component
Biological speciesHomo sapiens (human) / house mouse (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.34 Å
AuthorsGharpure A / Teng J / Zhuang Y / Noviello CM / Walsh RM / Cabuco R / Howard RJ / Zaveri NT / Lindahl E / Hibbs RE
Funding support United States, 6 items
OrganizationGrant numberCountry
Welch FoundationI-1812 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32GM008203 United States
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)T32DA07290 United States
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)DA042072 United States
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)DA037492 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS095899 United States
CitationJournal: Neuron / Year: 2019
Title: Agonist Selectivity and Ion Permeation in the α3β4 Ganglionic Nicotinic Receptor.
Authors: Anant Gharpure / Jinfeng Teng / Yuxuan Zhuang / Colleen M Noviello / Richard M Walsh / Rico Cabuco / Rebecca J Howard / Nurulain T Zaveri / Erik Lindahl / Ryan E Hibbs /
Abstract: Nicotinic acetylcholine receptors are pentameric ion channels that mediate fast chemical neurotransmission. The α3β4 nicotinic receptor subtype forms the principal relay between the central and ...Nicotinic acetylcholine receptors are pentameric ion channels that mediate fast chemical neurotransmission. The α3β4 nicotinic receptor subtype forms the principal relay between the central and peripheral nervous systems in the autonomic ganglia. This receptor is also expressed focally in brain areas that affect reward circuits and addiction. Here, we present structures of the α3β4 nicotinic receptor in lipidic and detergent environments, using functional reconstitution to define lipids appropriate for structural analysis. The structures of the receptor in complex with nicotine, as well as the α3β4-selective ligand AT-1001, complemented by molecular dynamics, suggest principles of agonist selectivity. The structures further reveal much of the architecture of the intracellular domain, where mutagenesis experiments and simulations define residues governing ion conductance.
History
DepositionJul 19, 2019-
Header (metadata) releaseAug 21, 2019-
Map releaseSep 11, 2019-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0275
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.0275
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6pv7
  • Surface level: 0.0275
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20487.png

Downloads & links

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Map

FileDownload / File: emd_20487.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.0275 / Movie #1: 0.0275
Minimum - Maximum-0.10608274 - 0.1819137
Average (Standard dev.)0.0002577802 (±0.0038265774)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 321.00003 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z321.000321.000321.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.1060.1820.000

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Supplemental data

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Half map: #1

Fileemd_20487_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_20487_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human alpha3beta4 nicotinic receptor in complex with Fab fragment...

EntireName: Human alpha3beta4 nicotinic receptor in complex with Fab fragments and bound to nicotine
Components
  • Complex: Human alpha3beta4 nicotinic receptor in complex with Fab fragments and bound to nicotine
    • Complex: Human alpha3beta4 nicotinic acetylcholine receptor
      • Complex: Neuronal acetylcholine receptor subunit alpha-3
        • Protein or peptide: Fusion protein of Neuronal acetylcholine receptor subunit alpha-3 and Soluble cytochrome b562
      • Complex: Neuronal acetylcholine receptor subunit beta-4
        • Protein or peptide: Fusion protein of Neuronal acetylcholine receptor subunit beta-4 and Soluble cytochrome b562
    • Complex: IgG2b/kappa antibody
      • Complex: IgG2b heavy chain
        • Protein or peptide: IgG2b Fab heavy chain
      • Complex: Kappa Fab light chain
        • Protein or peptide: Kappa Fab light chain
  • Ligand: (S)-3-(1-METHYLPYRROLIDIN-2-YL)PYRIDINE
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: SODIUM IONSodium
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: nonane
  • Ligand: water

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Supramolecule #1: Human alpha3beta4 nicotinic receptor in complex with Fab fragment...

SupramoleculeName: Human alpha3beta4 nicotinic receptor in complex with Fab fragments and bound to nicotine
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Details: Fab fragments generated by proteolytic cleavage of IgG antibody
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #2: Human alpha3beta4 nicotinic acetylcholine receptor

SupramoleculeName: Human alpha3beta4 nicotinic acetylcholine receptor / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2

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Supramolecule #3: Neuronal acetylcholine receptor subunit alpha-3

SupramoleculeName: Neuronal acetylcholine receptor subunit alpha-3 / type: complex / ID: 3 / Parent: 2 / Macromolecule list: #1
Recombinant expressionOrganism: Homo sapiens (human)

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Supramolecule #4: Neuronal acetylcholine receptor subunit beta-4

SupramoleculeName: Neuronal acetylcholine receptor subunit beta-4 / type: complex / ID: 4 / Parent: 2 / Macromolecule list: #2

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Supramolecule #5: IgG2b/kappa antibody

SupramoleculeName: IgG2b/kappa antibody / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #3-#4
Details: Fab fragment generated by proteolytic cleavage of IgG antibody

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Supramolecule #6: IgG2b heavy chain

SupramoleculeName: IgG2b heavy chain / type: complex / ID: 6 / Parent: 5 / Macromolecule list: #3

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Supramolecule #7: Kappa Fab light chain

SupramoleculeName: Kappa Fab light chain / type: complex / ID: 7 / Parent: 5 / Macromolecule list: #4

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Macromolecule #1: Fusion protein of Neuronal acetylcholine receptor subunit alpha-3...

MacromoleculeName: Fusion protein of Neuronal acetylcholine receptor subunit alpha-3 and Soluble cytochrome b562
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 60.087363 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: SEAEHRLFER LFEDYNEIIR PVANVSDPVI IHFEVSMSQL VKVDEVNQIM ETNLWLKQIW NDYKLKWNPS DYGGAEFMRV PAQKIWKPD IVLYNNAVGD FQVDDKTKAL LKYTGEVTWI PPAIFKSSCK IDVTYFPFDY QNCTMKFGSW SYDKAKIDLV L IGSSMNLK ...String:
SEAEHRLFER LFEDYNEIIR PVANVSDPVI IHFEVSMSQL VKVDEVNQIM ETNLWLKQIW NDYKLKWNPS DYGGAEFMRV PAQKIWKPD IVLYNNAVGD FQVDDKTKAL LKYTGEVTWI PPAIFKSSCK IDVTYFPFDY QNCTMKFGSW SYDKAKIDLV L IGSSMNLK DYWESGEWAI IKAPGYKHDI KYNCCEEIYP DITYSLYIRR LPLFYTINLI IPCLLISFLT VLVFYLPSDC GE KVTLCIS VLLSLTVFLL VITETIPSTS LVIPLIGEYL LFTMIFVTLS IVITVFVLNV HYRTPTTHTM PSWVKTVFLN LLP RVMFMT RPTSNEGNAQ KPRPLYGAEL SADLEDNWET LNDNLKVIEK ADNAAQVKDA LTKMRAAALD AQKATPPKLE DKSP DSPEM KDFRHGFDIL VGQIDDALKL ANEGKVKEAQ AAAEQLKTTR NAYIQKYLLS ALSPEIKEAI QSVKYIAENM KAQNE AKEI QDDWKYVAMV IDRIFLWVFT LVCILGTAGL FLQPLMARED A

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Macromolecule #2: Fusion protein of Neuronal acetylcholine receptor subunit beta-4 ...

MacromoleculeName: Fusion protein of Neuronal acetylcholine receptor subunit beta-4 and Soluble cytochrome b562
type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 61.337594 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: RVANAEEKLM DDLLNKTRYN NLIRPATSSS QLISIKLQLS LAQLISVNER EQIMTTNVWL KQEWTDYRLT WNSSRYEGVN ILRIPAKRI WLPDIVLYNN ADGTYEVSVY TNLIVRSNGS VLWLPPAIYK SACKIEVKYF PFDQQNCTLK FRSWTYDHTE I DMVLMTPT ...String:
RVANAEEKLM DDLLNKTRYN NLIRPATSSS QLISIKLQLS LAQLISVNER EQIMTTNVWL KQEWTDYRLT WNSSRYEGVN ILRIPAKRI WLPDIVLYNN ADGTYEVSVY TNLIVRSNGS VLWLPPAIYK SACKIEVKYF PFDQQNCTLK FRSWTYDHTE I DMVLMTPT ASMDDFTPSG EWDIVALPGR RTVNPQDPSY VDVTYDFIIK RKPLFYTINL IIPCVLTTLL AILVFYLPSD CG EKMTLCI SVLLALTFFL LLISKIVPPT SLDVPLIGKY LMFTMVLVTF SIVTSVCVLN VHHRSPSTHT MAPWVKRCFL HKL PTFLFM KRPGPDSSPA RAFPADLEDN WETLNDNLKV IEKADNAAQV KDALTKMRAA ALDAQKATPP KLEDKSPDSP EMKD FRHGF DILVGQIDDA LKLANEGKVK EAQAAAEQLK TTRNAYIQKY LSGRFRQDVQ EALEGVSFIA QHMKNDDEDQ SVVED WKYV AMVVDRLFLW VFMFVCVLGT VGLFLPPLFQ THAASEGPYA AQRDSAWSHP QFEK

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Macromolecule #3: IgG2b Fab heavy chain

MacromoleculeName: IgG2b Fab heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: house mouse (house mouse) / Strain: BALB/C
Molecular weightTheoretical: 23.396051 KDa
SequenceString: QVQLQQSGPE LVRPGVSVKI SCKGSGYTFT DYALYWVRQS HAKSLEWIGV INPYSGNTNY NQKFKGKATM TVDKSSSTAY MELARLTSD DSAIYYCASG YDGYPYTMDY WGPGASVTVS SAKTTPPSVY PLAPGCGDTT GSSVTLGCLV KGYFPESVTV T WNSGSLSS ...String:
QVQLQQSGPE LVRPGVSVKI SCKGSGYTFT DYALYWVRQS HAKSLEWIGV INPYSGNTNY NQKFKGKATM TVDKSSSTAY MELARLTSD DSAIYYCASG YDGYPYTMDY WGPGASVTVS SAKTTPPSVY PLAPGCGDTT GSSVTLGCLV KGYFPESVTV T WNSGSLSS SVHTFPALLQ SGLYTMSSSV TVPSSTWPSQ TVTCSVAHPA SSTTVDKKLE P

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Macromolecule #4: Kappa Fab light chain

MacromoleculeName: Kappa Fab light chain / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: house mouse (house mouse) / Strain: BALB/C
Molecular weightTheoretical: 23.401861 KDa
SequenceString: QIVLTQSPAI MSASPGEKVT MTCSASSSMT YMHWYQQKSG TSPKRWIYDT SQLASGVPAR FRGSGSGTSF SLTISSMEAE DAATYYCQQ WSSNPLTFGG GTKLELKRAD AAPTVSIFPP SSEQLTSGGA SVVCFLNNFY PKDINVKWKI DGSERQNGVL N SWTDQDSK ...String:
QIVLTQSPAI MSASPGEKVT MTCSASSSMT YMHWYQQKSG TSPKRWIYDT SQLASGVPAR FRGSGSGTSF SLTISSMEAE DAATYYCQQ WSSNPLTFGG GTKLELKRAD AAPTVSIFPP SSEQLTSGGA SVVCFLNNFY PKDINVKWKI DGSERQNGVL N SWTDQDSK DSTYSMSSTL TLTKDEYERH NSYTCEATHK TSTSPIVKSF NRNEC

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Macromolecule #8: (S)-3-(1-METHYLPYRROLIDIN-2-YL)PYRIDINE

MacromoleculeName: (S)-3-(1-METHYLPYRROLIDIN-2-YL)PYRIDINE / type: ligand / ID: 8 / Number of copies: 2 / Formula: NCT
Molecular weightTheoretical: 162.232 Da
Chemical component information

ChemComp-NCT:
(S)-3-(1-METHYLPYRROLIDIN-2-YL)PYRIDINE / alkaloid*YM / Nicotine

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Macromolecule #9: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 9 / Number of copies: 10 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #10: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 10 / Number of copies: 1
Molecular weightTheoretical: 22.99 Da

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Macromolecule #11: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 11 / Number of copies: 6 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Macromolecule #12: nonane

MacromoleculeName: nonane / type: ligand / ID: 12 / Number of copies: 1 / Formula: DD9
Molecular weightTheoretical: 128.255 Da
Chemical component information

ChemComp-DD9:
nonane / Nonane

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Macromolecule #13: water

MacromoleculeName: water / type: ligand / ID: 13 / Number of copies: 8 / 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

Concentration6 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris
150.0 mMNaClSodium chlorideSodium chloride
1.0 mMC9H15O6PTris(2-carboxyethyl)phosphine
1.0 mMC10H14N2Nicotine
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 4 second blot.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 46730 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 5053 / Average exposure time: 10.0 sec. / Average electron dose: 47.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 705401
CTF correctionSoftware - Name: Gctf
Startup modelType of model: OTHER / Details: De novo initial model from Relion
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.34 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 112737

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-6pv7:
Human alpha3beta4 nicotinic acetylcholine receptor in complex with nicotine

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