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- EMDB-7535: Structure of the 2alpha3beta stiochiometry of the human Alpha4Bet... -

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Entry
Database: EMDB / ID: EMD-7535
TitleStructure of the 2alpha3beta stiochiometry of the human Alpha4Beta2 nicotinic receptor
Map data
Sample
  • Complex: Complex of three Fab fragments with the 2alpha3beta stoichiometry of the human Alpha4Beta2 nicotinic receptor
    • Protein or peptide: Neuronal acetylcholine receptor subunit alpha-4
    • Protein or peptide: Neuronal acetylcholine receptor subunit beta-2
    • Protein or peptide: IgG1 Kappa Light Chain
    • Protein or peptide: IgG1 Heavy ChainImmunoglobulin G
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: (S)-3-(1-METHYLPYRROLIDIN-2-YL)PYRIDINE
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: SODIUM IONSodium
Function / homology
Function and homology information


vestibulocochlear nerve development / lateral geniculate nucleus development / regulation of circadian sleep/wake cycle, REM sleep / regulation of synaptic transmission, dopaminergic / quaternary ammonium group binding / synaptic transmission involved in micturition / Highly sodium permeable postsynaptic acetylcholine nicotinic receptors / Highly calcium permeable nicotinic acetylcholine receptors / optic nerve morphogenesis / central nervous system projection neuron axonogenesis ...vestibulocochlear nerve development / lateral geniculate nucleus development / regulation of circadian sleep/wake cycle, REM sleep / regulation of synaptic transmission, dopaminergic / quaternary ammonium group binding / synaptic transmission involved in micturition / Highly sodium permeable postsynaptic acetylcholine nicotinic receptors / Highly calcium permeable nicotinic acetylcholine receptors / optic nerve morphogenesis / central nervous system projection neuron axonogenesis / acetylcholine receptor activity / response to acetylcholine / Highly calcium permeable postsynaptic nicotinic acetylcholine receptors / regulation of dopamine metabolic process / acetylcholine-gated channel complex / cholinergic synapse / negative regulation of action potential / behavioral response to nicotine / positive regulation of dopamine secretion / acetylcholine-gated monoatomic cation-selective channel activity / synaptic transmission, cholinergic / postsynaptic specialization membrane / inhibitory postsynaptic potential / nervous system process / acetylcholine binding / acetylcholine receptor signaling pathway / regulation of synapse assembly / action potential / regulation of dendrite morphogenesis / regulation of dopamine secretion / heterocyclic compound binding / B cell activation / plasma membrane raft / social behavior / membrane depolarization / ligand-gated monoatomic ion channel activity / associative learning / smooth muscle contraction / monoatomic ion transport / positive regulation of B cell proliferation / sensory perception of pain / visual perception / regulation of membrane potential / response to cocaine / locomotory behavior / response to nicotine / learning / sensory perception of sound / visual learning / memory / cognition / calcium ion transport / presynaptic membrane / chemical synaptic transmission / postsynaptic membrane / response to ethanol / response to oxidative stress / response to hypoxia / neuron projection / external side of plasma membrane / DNA repair / dendrite / neuronal cell body / synapse / protein-containing complex binding / 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 / 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
Neuronal acetylcholine receptor subunit beta-2 / Neuronal acetylcholine receptor subunit alpha-4
Similarity search - Component
Biological speciesHomo sapiens (human) / house mouse (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsWalsh Jr RM / Roh SH / Gharpure A / Morales-Perez CL / Teng J / Hibbs RE
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS095899 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
CitationJournal: Nature / Year: 2018
Title: Structural principles of distinct assemblies of the human α4β2 nicotinic receptor.
Authors: Richard M Walsh / Soung-Hun Roh / Anant Gharpure / Claudio L Morales-Perez / Jinfeng Teng / Ryan E Hibbs /
Abstract: Fast chemical communication in the nervous system is mediated by neurotransmitter-gated ion channels. The prototypical member of this class of cell surface receptors is the cation-selective nicotinic ...Fast chemical communication in the nervous system is mediated by neurotransmitter-gated ion channels. The prototypical member of this class of cell surface receptors is the cation-selective nicotinic acetylcholine receptor. As with most ligand-gated ion channels, nicotinic receptors assemble as oligomers of subunits, usually as hetero-oligomers and often with variable stoichiometries . This intrinsic heterogeneity in protein composition provides fine tunability in channel properties, which is essential to brain function, but frustrates structural and biophysical characterization. The α4β2 subtype of the nicotinic acetylcholine receptor is the most abundant isoform in the human brain and is the principal target in nicotine addiction. This pentameric ligand-gated ion channel assembles in two stoichiometries of α- and β-subunits (2α:3β and 3α:2β). Both assemblies are functional and have distinct biophysical properties, and an imbalance in the ratio of assemblies is linked to both nicotine addiction and congenital epilepsy. Here we leverage cryo-electron microscopy to obtain structures of both receptor assemblies from a single sample. Antibody fragments specific to β2 were used to 'break' symmetry during particle alignment and to obtain high-resolution reconstructions of receptors of both stoichiometries in complex with nicotine. The results reveal principles of subunit assembly and the structural basis of the distinctive biophysical and pharmacological properties of the two different stoichiometries of this receptor.
History
DepositionMar 8, 2018-
Header (metadata) releaseApr 4, 2018-
Map releaseMay 2, 2018-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0327
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0327
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6cnj
  • Surface level: 0.0327
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7535_1.png

emd_7535_2.png

emd_7535_3.png

Downloads & links

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Map

FileDownload / File: emd_7535.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.0327 / Movie #1: 0.0327
Minimum - Maximum-0.11702618 - 0.20550187
Average (Standard dev.)0.0008976148 (±0.008614866)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 267.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z267.500267.500267.500
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS250250250
D min/max/mean-0.1170.2060.001

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Supplemental data

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Half map: #1

Fileemd_7535_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_7535_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 : Complex of three Fab fragments with the 2alpha3beta stoichiometry...

EntireName: Complex of three Fab fragments with the 2alpha3beta stoichiometry of the human Alpha4Beta2 nicotinic receptor
Components
  • Complex: Complex of three Fab fragments with the 2alpha3beta stoichiometry of the human Alpha4Beta2 nicotinic receptor
    • Protein or peptide: Neuronal acetylcholine receptor subunit alpha-4
    • Protein or peptide: Neuronal acetylcholine receptor subunit beta-2
    • Protein or peptide: IgG1 Kappa Light Chain
    • Protein or peptide: IgG1 Heavy ChainImmunoglobulin G
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: (S)-3-(1-METHYLPYRROLIDIN-2-YL)PYRIDINE
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: SODIUM IONSodium

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Supramolecule #1: Complex of three Fab fragments with the 2alpha3beta stoichiometry...

SupramoleculeName: Complex of three Fab fragments with the 2alpha3beta stoichiometry of the human Alpha4Beta2 nicotinic receptor
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Details: Fab fragmented generated by proteolytic cleavage of IgG antibody
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 344 KDa

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Macromolecule #1: Neuronal acetylcholine receptor subunit alpha-4

MacromoleculeName: Neuronal acetylcholine receptor subunit alpha-4 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 44.862367 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: SSHVETRAHA EERLLKKLFS GYNKWSRPVA NISDVVLVRF GLSIAQLIDV DEKNQMMTTN VWVKQEWHDY KLRWDPADYE NVTSIRIPS ELIWRPDIVL YNNADGDFAV THLTKAHLFH DGRVQWTPPA IYKSSCSIDV TFFPFDQQNC TMKFGSWTYD K AKIDLVNM ...String:
SSHVETRAHA EERLLKKLFS GYNKWSRPVA NISDVVLVRF GLSIAQLIDV DEKNQMMTTN VWVKQEWHDY KLRWDPADYE NVTSIRIPS ELIWRPDIVL YNNADGDFAV THLTKAHLFH DGRVQWTPPA IYKSSCSIDV TFFPFDQQNC TMKFGSWTYD K AKIDLVNM HSRVDQLDFW ESGEWVIVDA VGTYNTRKYE CCAEIYPDIT YAFVIRRLPL FYTINLIIPC LLISCLTVLV FY LPSECGE KITLCISVLL SLTVFLLLIT EIIPSTSLVI PLIGEYLLFT MIFVTLSIVI TVFVLNVHHR SPRTHTMPTW VRR VFLDIV PRLLLMKRPS VVDTDFERSV KEDWKYVAMV IDRIFLWMFI IVCLLGTVGL FLPPWLAGMI

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Macromolecule #2: Neuronal acetylcholine receptor subunit beta-2

MacromoleculeName: Neuronal acetylcholine receptor subunit beta-2 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 46.748863 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: TDTEERLVEH LLDPSRYNKL IRPATNGSEL VTVQLMVSLA QLISVHEREQ IMTTNVWLTQ EWEDYRLTWK PEEFDNMKKV RLPSKHIWL PDVVLYNNAD GMYEVSFYSN AVVSYDGSIF WLPPAIYKSA CKIEVKHFPF DQQNCTMKFR SWTYDRTEID L VLKSEVAS ...String:
TDTEERLVEH LLDPSRYNKL IRPATNGSEL VTVQLMVSLA QLISVHEREQ IMTTNVWLTQ EWEDYRLTWK PEEFDNMKKV RLPSKHIWL PDVVLYNNAD GMYEVSFYSN AVVSYDGSIF WLPPAIYKSA CKIEVKHFPF DQQNCTMKFR SWTYDRTEID L VLKSEVAS LDDFTPSGEW DIVALPGRRN ENPDDSTYVD ITYDFIIRRK PLFYTINLII PCVLITSLAI LVFYLPSDCG EK MTLCISV LLALTVFLLL ISKIVPPTSL DVPLVGKYLM FTMVLVTFSI VTSVCVLNVH HRSPTTHTMA PWVKVVFLEK LPA LLFMQQ PRHHDDDQER SVSEDWKYVA MVIDRLFLWI FVFVCVFGTI GMFLQPLFQN YTTTTFLHSD HSAPSSKSAW SHPQ FEK

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Macromolecule #3: IgG1 Kappa Light Chain

MacromoleculeName: IgG1 Kappa Light Chain / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: house mouse (house mouse) / Strain: BALB/c
Molecular weightTheoretical: 26.378596 KDa
SequenceString: MKLPVRLLVL MFWIPASSSD VLMTQTPLSL PVSLGDQASI SCRSSQSIVH SNGNTYLEWY LQKPGQSPKL LIYKVSNRFS GVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSHVP WTFGGGTKLE IKRADAAPTV SIFPPSSEQL TSGGASVVCF L NNFYPKDI ...String:
MKLPVRLLVL MFWIPASSSD VLMTQTPLSL PVSLGDQASI SCRSSQSIVH SNGNTYLEWY LQKPGQSPKL LIYKVSNRFS GVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSHVP WTFGGGTKLE IKRADAAPTV SIFPPSSEQL TSGGASVVCF L NNFYPKDI NVKWKIDGSE RQNGVLNSWT DQDSKDSTYS MSSTLTLTKD EYERHNSYTC EATHKTSTSP IVKSFNRNEC

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Macromolecule #4: IgG1 Heavy Chain

MacromoleculeName: IgG1 Heavy Chain / type: protein_or_peptide / ID: 4 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: house mouse (house mouse) / Strain: BALB/c
Molecular weightTheoretical: 51.195668 KDa
SequenceString: MEWTWVFLFL LSVTAGVHSQ VQLQQSGAEV MKPGASVKIS CKGTGYTFSS YWIEWVKQRP GHGLERIGEI LPGSGSTNYN EKFRGKATF TADKSSKTAY MQLSSLTSED SAVYYCARYL PYYYAMDYWG QGTSVTVSSA KTTPPSVYPL APGSAAQTNS M VTLGCLVK ...String:
MEWTWVFLFL LSVTAGVHSQ VQLQQSGAEV MKPGASVKIS CKGTGYTFSS YWIEWVKQRP GHGLERIGEI LPGSGSTNYN EKFRGKATF TADKSSKTAY MQLSSLTSED SAVYYCARYL PYYYAMDYWG QGTSVTVSSA KTTPPSVYPL APGSAAQTNS M VTLGCLVK GYFPEPVTVT WNSGSLSSGV HTFPAVLQSD LYTLSSSVTV PSSTWPSETV TCNVAHPASS TKVDKKIVPR DC GCKPCIC TVPEVSSVFI FPPKPKDVLT ITLTPKVTCV VVDISKDDPE VQFSWFVDDV EVHTAQTQPR EEQFNSTFRS VSE LPIMHQ DWLNGKEFKC RVNSAAFPAP IEKTISKTKG RPKAPQVYTI PPPKEQMAKD KVSLTCMITD FFPEDITVEW QWNG QPAEN YKNTQPIMDT DGSYFVYSKL NVQKSNWEAG NTFTCSVLHE GLHNHHTEKS LSHSPGK

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Macromolecule #6: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 6 / Number of copies: 2 / 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 #7: (S)-3-(1-METHYLPYRROLIDIN-2-YL)PYRIDINE

MacromoleculeName: (S)-3-(1-METHYLPYRROLIDIN-2-YL)PYRIDINE / type: ligand / ID: 7 / 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 #8: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 8 / Number of copies: 10 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #9: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 9 / Number of copies: 1
Molecular weightTheoretical: 22.99 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

Concentration6 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
20.0 mMC4H11NO3Trizma base
1.0 mMC24H46O11n-Dodecyl beta-D-maltoside
0.2 mMC31H50O4Cholesterol Hemisuccinate
1.0 mMC10H14N2Nicotine
1.0 mMTCEP (2-carboxyethyl)phosphine) hydrocloride
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 39.0 kPa
Details: Sample was glow discharged at 30mA for 80 seconds using a PELCO easiGLow
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 4 second blot time.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 46730 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 0.5 µm
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Lower energy threshold: -10 eV / Energy filter - Upper energy threshold: 10 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 80.0 K / Max: 90.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 2 / Number real images: 5166 / Average exposure time: 15.0 sec. / Average electron dose: 75.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 649773
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: OTHER / Details: Starting model made in RELION 2.1
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 8 / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1)
Final reconstructionNumber classes used: 4 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 263695
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-6cnj:
Structure of the 2alpha3beta stiochiometry of the human Alpha4Beta2 nicotinic receptor

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