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- PDB-6cnk: Structure of the 3alpha2beta stiochiometry of the human Alpha4Bet... -

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Basic information

Entry
Database: PDB / ID: 6cnk
TitleStructure of the 3alpha2beta stiochiometry of the human Alpha4Beta2 nicotinic receptor
Components
  • (Neuronal acetylcholine receptor subunit ...) x 2
  • IgG1 Heavy ChainImmunoglobulin G
  • IgG1 Kappa Light Chain
KeywordsTRANSPORT 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 / 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 / Acetylcholine Binding Protein; Chain: A, / Neurotransmitter-gated ion-channel ligand-binding domain / 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 ...Nicotinic acetylcholine receptor / Acetylcholine Binding Protein; Chain: A, / Neurotransmitter-gated ion-channel ligand-binding domain / 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 / Distorted Sandwich / Immunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
(S)-3-(1-METHYLPYRROLIDIN-2-YL)PYRIDINE / CHOLESTEROL HEMISUCCINATE / Neuronal acetylcholine receptor subunit beta-2 / Neuronal acetylcholine receptor subunit alpha-4
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsWalsh Jr, R.M. / Roh, S.H. / Gharpure, A. / Morales-Perez, C.L. / Hibbs, R.E.
Funding support United States, 3items
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)DA037492 United States
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)DA042072 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, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 2, 2018Provider: repository / Type: Initial release
Revision 1.1May 9, 2018Group: Data collection / Structure summary / Category: audit_author / Item: _audit_author.name
Revision 1.2May 16, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.3May 30, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.4Jul 18, 2018Group: Data collection / Category: em_imaging_optics / Item: _em_imaging_optics.energyfilter_name
Revision 1.5Nov 20, 2019Group: Data collection / Database references / Category: em_imaging_optics / pdbx_database_related
Item: _em_imaging_optics.energyfilter_name / _pdbx_database_related.db_id
Revision 1.6Dec 11, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.7Dec 18, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB
Revision 2.0Jul 29, 2020Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / em_entity_assembly / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / pdbx_unobs_or_zero_occ_atoms / struct_asym / struct_conn / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _chem_comp.name / _chem_comp.pdbx_synonyms / _chem_comp.type / _em_entity_assembly.entity_id_list / _entity.formula_weight / _entity.pdbx_description / _entity.pdbx_number_of_molecules / _entity.src_method / _entity.type / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_unobs_or_zero_occ_atoms.label_asym_id / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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Structure visualization

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Assembly

Deposited unit
A: Neuronal acetylcholine receptor subunit alpha-4
B: Neuronal acetylcholine receptor subunit alpha-4
C: Neuronal acetylcholine receptor subunit beta-2
D: Neuronal acetylcholine receptor subunit alpha-4
E: Neuronal acetylcholine receptor subunit beta-2
F: IgG1 Kappa Light Chain
G: IgG1 Heavy Chain
J: IgG1 Kappa Light Chain
K: IgG1 Heavy Chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)390,44728
Polymers383,2339
Non-polymers7,21419
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Gel Filtration used to produce homogeneous sample of alpha4Beta2:Fab complex.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Neuronal acetylcholine receptor subunit ... , 2 types, 5 molecules ABDCE

#1: Protein Neuronal acetylcholine receptor subunit alpha-4


Mass: 44862.367 Da / Num. of mol.: 3
Mutation: Glu-Arg linker was inserted in the MX-M4 junction, between Phe559-Ser560 in the alpha4 subunit,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 Neuronal acetylcholine receptor subunit beta-2


Mass: 46748.863 Da / Num. of mol.: 2
Mutation: Glu-Arg linker was inserted in the MX-M4 junction between Gln420-Ser421 in the beta 2 subunit.,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, 4 molecules FJGK

#3: Antibody IgG1 Kappa Light Chain


Mass: 26378.596 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Plasmid details: From generated Hybridoma / Strain: BALB/c
#4: Antibody IgG1 Heavy Chain / Immunoglobulin G


Mass: 51195.668 Da / Num. of mol.: 2 / 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 beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 2 / Source method: obtained synthetically
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}}LINUCSPDB-CARE
#6: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 3 types, 14 molecules

#7: Chemical
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C31H50O4
#8: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#9: Chemical ChemComp-NCT / (S)-3-(1-METHYLPYRROLIDIN-2-YL)PYRIDINE / (S)-(-)-NICOTINE / 3-[(2S)-1-METHYL-2-PYRROLIDINYL] PYRIDINE / Nicotine


Mass: 162.232 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H14N2 / Comment: alkaloid*YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Complex of two Fab fragments with the 3alpha2beta 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
Molecular weightValue: 0.294 MDa
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaClSodium chloride1
220 mMTrizma baseC4H11NO31
31 mMn-Dodecyl beta-D-maltosideC24H46O111
40.2 mMCholesterol HemisuccinateC31H50O41
51 mMTCEP (2-carboxyethyl)phosphine) hydrocloride1
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 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
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 4 second blot time

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / 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 holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 K / Temperature (min): 80 K
Image recordingAverage 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 opticsEnergyfilter name: GIF Quantum LS / Energyfilter upper: 10 eV / Energyfilter lower: -10 eV

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Processing

EM software
IDNameVersionCategory
1RELION2.1particle selection
2EPUimage acquisition
4Gctf1.06CTF correction
7Coot0.8.9model fitting
9RELION2.1initial Euler assignment
10RELION2.1final Euler assignment
11RELION2.1classification
12RELION2.13D reconstruction
13PHENIX1.13-2988model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 649773
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 139551 / Algorithm: BACK PROJECTION / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
RefinementHighest resolution: 3.7 Å

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