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Yorodumi- PDB-6cnj: Structure of the 2alpha3beta stiochiometry of the human Alpha4Bet... -
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-Basic information
Entry | Database: PDB / ID: 6cnj | ||||||||||||
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Title | Structure of the 2alpha3beta stiochiometry of the human Alpha4Beta2 nicotinic receptor | ||||||||||||
Components |
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Keywords | TRANSPORT PROTEIN / Ligand-gated ion channel / Acetylcholine receptor / Cys-loop Receptor / MEMBRANE PROTEIN | ||||||||||||
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 / synaptic transmission involved in micturition / quaternary ammonium group binding / 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 / synaptic transmission involved in micturition / quaternary ammonium group binding / Highly sodium permeable postsynaptic acetylcholine nicotinic receptors / Highly calcium permeable nicotinic acetylcholine receptors / optic nerve morphogenesis / central nervous system projection neuron axonogenesis / Highly calcium permeable postsynaptic nicotinic acetylcholine receptors / acetylcholine receptor activity / response to acetylcholine / cholinergic synapse / acetylcholine-gated channel complex / negative regulation of action potential / positive regulation of dopamine secretion / regulation of dopamine metabolic process / behavioral response to nicotine / acetylcholine-gated monoatomic cation-selective channel activity / inhibitory postsynaptic potential / nervous system process / synaptic transmission, cholinergic / acetylcholine binding / acetylcholine receptor signaling pathway / postsynaptic specialization membrane / heterocyclic compound binding / regulation of synapse assembly / regulation of dendrite morphogenesis / action potential / regulation of dopamine secretion / plasma membrane raft / B cell activation / associative learning / membrane depolarization / social behavior / ligand-gated monoatomic ion channel activity / smooth muscle contraction / monoatomic ion transport / positive regulation of B cell proliferation / visual perception / sensory perception of pain / regulation of membrane potential / learning / response to cocaine / cognition / locomotory behavior / response to nicotine / sensory perception of sound / visual learning / memory / 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 / neuronal cell body / dendrite / synapse / protein-containing complex binding / signal transduction / membrane / plasma membrane Similarity search - Function | ||||||||||||
Biological species | Homo sapiens (human) Mus musculus (house mouse) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å | ||||||||||||
Authors | Walsh Jr, R.M. / Roh, S.H. / Gharpure, A. / Morales-Perez, C.L. / Teng, J. / Hibbs, R.E. | ||||||||||||
Funding support | United States, 3items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6cnj.cif.gz | 564.3 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6cnj.ent.gz | 463.8 KB | Display | PDB format |
PDBx/mmJSON format | 6cnj.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6cnj_validation.pdf.gz | 1.8 MB | Display | wwPDB validaton report |
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Full document | 6cnj_full_validation.pdf.gz | 1.9 MB | Display | |
Data in XML | 6cnj_validation.xml.gz | 102.4 KB | Display | |
Data in CIF | 6cnj_validation.cif.gz | 145.1 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/cn/6cnj ftp://data.pdbj.org/pub/pdb/validation_reports/cn/6cnj | HTTPS FTP |
-Related structure data
Related structure data | 7535MC 7536C 6cnkC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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-Components
-Neuronal acetylcholine receptor subunit ... , 2 types, 5 molecules ADBCE
#1: Protein | Mass: 44862.367 Da / Num. of mol.: 2 Mutation: Glu-Arg linker was inserted in the MX-M4 junction, between Phe559-Ser560 in the alpha4 subunit Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: CHRNA4, NACRA4 / Plasmid: pEZT-BM / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / Variant (production host): GnTI- / References: UniProt: P43681 #2: Protein | Mass: 46748.863 Da / Num. of mol.: 3 Mutation: Glu-Arg linker was inserted in the MX-M4 junction between Gln420-Ser421 in the beta 2 subunit. Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: CHRNB2 / Plasmid: pEZT-BM / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / Variant (production host): GnTI- / References: UniProt: P17787 |
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-Antibody , 2 types, 6 molecules FHJGIK
#3: Antibody | Mass: 26378.596 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Plasmid details: From generated Hybridoma / Strain: BALB/c #4: Antibody | Mass: 51195.668 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Plasmid details: From generated Hybridoma / Strain: BALB/c |
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-Sugars , 2 types, 5 molecules
#5: Polysaccharide | #6: Sugar | |
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-Non-polymers , 3 types, 13 molecules
#7: Chemical | #8: Chemical | ChemComp-Y01 / #9: Chemical | ChemComp-NA / | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Complex of three Fab fragments with the 2alpha3beta stoichiometry of the human Alpha4Beta2 nicotinic receptor Type: COMPLEX Details: Fab fragmented generated by proteolytic cleavage of IgG antibody Entity ID: #1-#4 / Source: MULTIPLE SOURCES | |||||||||||||||||||||||||||||||||||
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Molecular weight | Value: 0.344 MDa | |||||||||||||||||||||||||||||||||||
Source (natural) | Organism: Homo sapiens (human) | |||||||||||||||||||||||||||||||||||
Source (recombinant) | Organism: Homo sapiens (human) / Strain: HEK293 GnT1- / Plasmid: pEZT-BM | |||||||||||||||||||||||||||||||||||
Buffer solution | pH: 7.4 | |||||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||||||||||||||||||||||
Specimen support | Details: Sample was glow discharged at 30mA for 80 seconds using a PELCO easiGLow Grid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3 | |||||||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 4 second blot time |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Calibrated magnification: 46730 X / Nominal defocus max: 4500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 K / Temperature (min): 80 K |
Image recording | Average exposure time: 15 sec. / Electron dose: 75 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 5166 |
EM imaging optics | Energyfilter name: GIF Quantum LS / Energyfilter upper: 10 eV / Energyfilter lower: -10 eV |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 649773 | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 263695 / Algorithm: BACK PROJECTION / Num. of class averages: 4 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: AB INITIO MODEL / Space: REAL | ||||||||||||||||||||||||||||||||||||||||
Refinement | Highest resolution: 3.4 Å |