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- PDB-6pv8: Human alpha3beta4 nicotinic acetylcholine receptor in complex wit... -

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Basic information

Entry
Database: PDB / ID: 6pv8
TitleHuman alpha3beta4 nicotinic acetylcholine receptor in complex with AT-1001
Components
  • (Fusion protein of Neuronal acetylcholine receptor subunit ...) x 2
  • IgG2b Fab heavy chain
  • Kappa Fab light chain
KeywordsMEMBRANE PROTEIN / Ligand-gated ion channel / Nicotinic acetylcholine receptor / Cys-loop receptor
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 / 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 / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 ...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 / 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 / Distorted Sandwich / Mainly Beta
Similarity search - Domain/homology
Chem-P1M / CHOLESTEROL HEMISUCCINATE / Soluble cytochrome b562 / Neuronal acetylcholine receptor subunit beta-4 / Neuronal acetylcholine receptor subunit alpha-3
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli O11 (bacteria)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.87 Å
AuthorsGharpure, A. / Teng, J. / Zhuang, Y. / Noviello, C.M. / Walsh, R.M. / Cabuco, R. / Howard, R.J. / Zaveri, N.T. / Lindahl, E. / Hibbs, R.E.
Funding support United States, 6items
OrganizationGrant numberCountry
Welch FoundationI-1812 United States
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)T32DA07290 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)DA037492 United States
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)DA042072 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, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 11, 2019Provider: repository / Type: Initial release
Revision 1.1Sep 18, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Nov 20, 2019Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.3Dec 11, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
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 / 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.type / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_unobs_or_zero_occ_atoms.label_asym_id / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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Structure visualization

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Assembly

Deposited unit
A: Fusion protein of Neuronal acetylcholine receptor subunit alpha-3 and Soluble cytochrome b562
B: Fusion protein of Neuronal acetylcholine receptor subunit beta-4 and Soluble cytochrome b562
C: Fusion protein of Neuronal acetylcholine receptor subunit beta-4 and Soluble cytochrome b562
D: Fusion protein of Neuronal acetylcholine receptor subunit alpha-3 and Soluble cytochrome b562
E: Fusion protein of Neuronal acetylcholine receptor subunit beta-4 and Soluble cytochrome b562
F: IgG2b Fab heavy chain
G: Kappa Fab light chain
H: IgG2b Fab heavy chain
I: Kappa Fab light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)411,78238
Polymers397,7839
Non-polymers13,99929
Water1448
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Fusion protein of Neuronal acetylcholine receptor subunit ... , 2 types, 5 molecules ADBCE

#1: Protein Fusion protein of Neuronal acetylcholine receptor subunit alpha-3 and Soluble cytochrome b562 /


Mass: 60087.363 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Escherichia coli O11 (bacteria)
Gene: CHRNA3, NACHRA3, CWJ32_12820 / Plasmid: pEZT-BM / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / Variant (production host): GNTI- / References: UniProt: P32297, UniProt: A0A3W4NZ06
#2: Protein Fusion protein of Neuronal acetylcholine receptor subunit beta-4 and Soluble cytochrome b562 /


Mass: 61337.594 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Escherichia coli O11 (bacteria)
Gene: CHRNB4, CWJ32_12820 / Plasmid: pEZT-BM / Production host: Homo sapiens (human) / Variant (production host): GNTI- / References: UniProt: P30926, UniProt: A0A3W4NZ06

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Antibody , 2 types, 4 molecules FHGI

#3: Antibody IgG2b Fab heavy chain


Mass: 23396.051 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Strain: BALB/C
#4: Antibody Kappa Fab light chain


Mass: 23401.861 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Strain: BALB/C

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Sugars , 4 types, 16 molecules

#5: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#6: Polysaccharide
alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 910.823 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3/a4-b1_b4-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}}}LINUCSPDB-CARE
#7: 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.: 3
Source method: isolated from a genetically manipulated source
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
#10: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
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 , 4 types, 21 molecules

#8: Chemical ChemComp-P1M / (3-endo)-N-(2-bromophenyl)-9-methyl-9-azabicyclo[3.3.1]nonan-3-amine


Mass: 309.245 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C15H21BrN2 / Feature type: SUBJECT OF INVESTIGATION
#9: Chemical
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


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


Mass: 22.990 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: Na
#12: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Human alpha3beta4 nicotinic receptor in complex with Fab fragments and bound to AT-1001COMPLEXFab fragments generated by proteolytic cleavage of IgG antibody#1-#40MULTIPLE SOURCES
2Human alpha3beta4 nicotinic acetylcholine receptorCOMPLEX#1-#21RECOMBINANT
3Neuronal acetylcholine receptor subunit alpha-3COMPLEX#12RECOMBINANT
4Neuronal acetylcholine receptor subunit beta-4COMPLEX#22RECOMBINANT
5IgG2b/kappa antibodyCOMPLEXFab fragment generated by proteolytic cleavage of IgG antibody#3-#41NATURAL
6IgG2b heavy chainCOMPLEX#35NATURAL
7Kappa Fab light chainCOMPLEX#45NATURAL
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
31NO
41NO
51NO
61NO
71NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
21Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
21Mus musculus (house mouse)10090
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
2150 mMSodium chlorideNaClSodium chloride1
31 mMTris(2-carboxyethyl)phosphine (TCEP)C9H15O6P1
40.05 mMAT-1001C15H22BrN21
51 mMDodecyl maltosideC24H46O111
60.2 mMCholesteryl hemisuccinateC31H50O41
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 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

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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: 59523 X / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 48 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 3008
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 20

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Processing

EM software
IDNameCategory
1RELIONparticle selection
2EPUimage acquisition
4GctfCTF correction
7Cootmodel fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
12RELION3D reconstruction
19PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 548045
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.87 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 93080 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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