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- EMDB-40753: The 2alpha3beta stoichiometry of full-length human alpha4beta2 ni... -

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Basic information

Entry
Database: EMDB / ID: EMD-40753
TitleThe 2alpha3beta stoichiometry of full-length human alpha4beta2 nicotinic acetylcholine receptor in complex with acetylcholine
Map data
Sample
  • Complex: A complex of three Fab fragments with the 2alpha3beta stoichiometry of the full-length human alpha4beta2 nicotinic receptor bound to acetylcholine
    • 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 Chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: ACETYLCHOLINE
  • Ligand: N-OCTANE
  • Ligand: SODIUM ION
  • Ligand: water
Keywordscys-loop ligand-gated pentameric ion channels / cation-selective channel / acetylcholine / TRANSPORT 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 / sensory perception of pain / visual perception / regulation of membrane potential / learning / response to cocaine / locomotory behavior / response to nicotine / 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 / neuronal cell body / dendrite / 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) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsKang G / Hibbs RE
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)DA047848 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)NS120496 United States
CitationJournal: Br J Pharmacol / Year: 2024
Title: Structural bases for stoichiometry-selective calcium potentiation of a neuronal nicotinic receptor.
Authors: Simone Mazzaferro / Guipeun Kang / Kathiresan Natarajan / Ryan E Hibbs / Steven M Sine /
Abstract: BACKGROUND AND PURPOSE: α4β2 nicotinic acetylcholine (nACh) receptors assemble in two stoichiometric forms, one of which is potentiated by calcium. The sites of calcium binding that underpin potentiation are not known.
EXPERIMENTAL APPROACH: To identify calcium binding sites, we applied cryo-electron microscopy (cryo-EM) and molecular dynamics (MD) simulations to each stoichiometric form of the α4β2 nACh receptor ...EXPERIMENTAL APPROACH: To identify calcium binding sites, we applied cryo-electron microscopy (cryo-EM) and molecular dynamics (MD) simulations to each stoichiometric form of the α4β2 nACh receptor in the presence of calcium ions. To test whether the identified calcium sites are linked to potentiation, we generated mutants of anionic residues at the sites, expressed wild type and mutant receptors in clonal mammalian fibroblasts, and recorded ACh-elicited single-channel currents with or without calcium.
KEY RESULTS: Both cryo-EM and MD simulations show calcium bound to a site between the extracellular and transmembrane domains of each α4 subunit (ECD-TMD site). Substituting alanine for anionic ...KEY RESULTS: Both cryo-EM and MD simulations show calcium bound to a site between the extracellular and transmembrane domains of each α4 subunit (ECD-TMD site). Substituting alanine for anionic residues at the ECD-TMD site abolishes stoichiometry-selective calcium potentiation, as monitored by single-channel patch clamp electrophysiology. Additionally, MD simulation reveals calcium association at subunit interfaces within the extracellular domain. Substituting alanine for anionic residues at the ECD sites reduces or abolishes stoichiometry-selective calcium potentiation.
CONCLUSIONS AND IMPLICATIONS: Stoichiometry-selective calcium potentiation of the α4β2 nACh receptor is achieved by calcium association with topographically distinct sites framed by anionic ...CONCLUSIONS AND IMPLICATIONS: Stoichiometry-selective calcium potentiation of the α4β2 nACh receptor is achieved by calcium association with topographically distinct sites framed by anionic residues within the α4 subunit and between the α4 and β2 subunits. Stoichiometry-selective calcium potentiation could result from the greater number of calcium sites in the stoichiometric form with three rather than two α4 subunits. The results are relevant to modulation of signalling via α4β2 nACh receptors in physiological and pathophysiological conditions.
History
DepositionMay 9, 2023-
Header (metadata) releaseFeb 7, 2024-
Map releaseFeb 7, 2024-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40753.png

Downloads & links

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Map

FileDownload / File: emd_40753.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 300 pix.
= 323.7 Å		1.08 Å/pix.
x 300 pix.
= 323.7 Å		1.08 Å/pix.
x 300 pix.
= 323.7 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.079 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.11775969 - 0.23435542
Average (Standard dev.)0.00043243935 (±0.0052806875)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 323.7 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_40753_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_40753_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : A complex of three Fab fragments with the 2alpha3beta stoichiomet...

EntireName: A complex of three Fab fragments with the 2alpha3beta stoichiometry of the full-length human alpha4beta2 nicotinic receptor bound to acetylcholine
Components
  • Complex: A complex of three Fab fragments with the 2alpha3beta stoichiometry of the full-length human alpha4beta2 nicotinic receptor bound to acetylcholine
    • 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 Chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: ACETYLCHOLINE
  • Ligand: N-OCTANE
  • Ligand: SODIUM ION
  • Ligand: water

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Supramolecule #1: A complex of three Fab fragments with the 2alpha3beta stoichiomet...

SupramoleculeName: A complex of three Fab fragments with the 2alpha3beta stoichiometry of the full-length human alpha4beta2 nicotinic receptor bound to acetylcholine
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)

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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: 67.411773 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 VVKDNCRRLI ESMHKMASAP RFWPEPEGEP PATSGTQSLH PPSPSFCVPL DVPAEPGPSC KSPS DQLPP QQPLEAEKAS PHPSPGPCRP PHGTQAPGLA KARSLSVQHM SSPGEAVEGG VRCRSRSIQY CVPRDDAAPE ADGQA AGAL ASRNTHSAEL PPPDQPSPCK CTCKKEPSSV SPSATVKTRS TKAPPPHLPL SPALTRAVEG VQYIADHLKA EDTDFS VKE DWKYVAMVID RIFLWMFIIV CLLGTVGLFL PPWLAGMI

UniProtKB: Neuronal acetylcholine receptor subunit alpha-4

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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: 55.649953 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 PRHHCARQRL RLRRRQRERE GAGALFFREA PGADSCTCFV NRASVQGLAG AFGAEPAPVA GPGRSGEPCG CGLR EAVDG VRFIADHMRS EDDDQSVSED WKYVAMVIDR LFLWIFVFVC VFGTIGMFLQ PLFQNYTTTT FLHSDHSAPS SKSAW SHPQ FEK

UniProtKB: Neuronal acetylcholine receptor subunit beta-2

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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: Mus musculus (house mouse)
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: Mus musculus (house mouse)
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

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Macromolecule #7: ACETYLCHOLINE

MacromoleculeName: ACETYLCHOLINE / type: ligand / ID: 7 / Number of copies: 2 / Formula: ACH
Molecular weightTheoretical: 146.207 Da
Chemical component information

ChemComp-ACH:
ACETYLCHOLINE / neurotransmitter*YM

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Macromolecule #8: N-OCTANE

MacromoleculeName: N-OCTANE / type: ligand / ID: 8 / Number of copies: 1 / Formula: OCT
Molecular weightTheoretical: 114.229 Da
Chemical component information

ChemComp-OCT:
N-OCTANE

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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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Macromolecule #10: water

MacromoleculeName: water / type: ligand / ID: 10 / Number of copies: 7 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
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

BufferpH: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6cnj

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: PHENIX (ver. 1.20.1_4487) / Number images used: 372621
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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