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- PDB-7tnj: Complex NNNN of AMPA-subtype iGluR GluA2 in complex with auxiliar... -

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Basic information

Entry
Database: PDB / ID: 7tnj
TitleComplex NNNN of AMPA-subtype iGluR GluA2 in complex with auxiliary subunit gamma2 (Stargazin) at low glutamate concentration (20 uM) in the presence of cyclothiazide (100 uM)
ComponentsIsoform Flip of Glutamate receptor 2,Voltage-dependent calcium channel gamma-3 subunit chimera
KeywordsMEMBRANE PROTEIN / AMPA / iGluR / Stargazin / cryo-EM / complex / agonist / positive allosteric modulator / subconductance level / opening / gating / glutamate
Function / homology
Function and homology information


: / : / : / neurotransmitter receptor transport, postsynaptic endosome to lysosome / LGI-ADAM interactions / Trafficking of AMPA receptors / regulation of AMPA receptor activity / neurotransmitter receptor internalization / channel regulator activity / postsynaptic neurotransmitter receptor diffusion trapping ...: / : / : / neurotransmitter receptor transport, postsynaptic endosome to lysosome / LGI-ADAM interactions / Trafficking of AMPA receptors / regulation of AMPA receptor activity / neurotransmitter receptor internalization / channel regulator activity / postsynaptic neurotransmitter receptor diffusion trapping / neurotransmitter receptor localization to postsynaptic specialization membrane / Activation of AMPA receptors / response to lithium ion / perisynaptic space / transmission of nerve impulse / AMPA glutamate receptor activity / plasma membrane => GO:0005886 / Trafficking of GluR2-containing AMPA receptors / immunoglobulin binding / ligand-gated monoatomic ion channel activity / AMPA glutamate receptor complex / kainate selective glutamate receptor activity / excitatory synapse / ionotropic glutamate receptor complex / extracellularly glutamate-gated ion channel activity / asymmetric synapse / protein targeting / regulation of receptor recycling / Unblocking of NMDA receptors, glutamate binding and activation / voltage-gated calcium channel activity / glutamate receptor binding / positive regulation of synaptic transmission / glutamate-gated receptor activity / response to fungicide / regulation of synaptic transmission, glutamatergic / somatodendritic compartment / ionotropic glutamate receptor binding / ionotropic glutamate receptor signaling pathway / dendrite cytoplasm / cytoskeletal protein binding / positive regulation of synaptic transmission, glutamatergic / SNARE binding / dendritic shaft / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / synaptic membrane / PDZ domain binding / postsynaptic density membrane / protein tetramerization / Schaffer collateral - CA1 synapse / establishment of protein localization / protein localization / terminal bouton / receptor internalization / synaptic vesicle membrane / synaptic vesicle / signaling receptor activity / presynaptic membrane / amyloid-beta binding / ATPase binding / growth cone / perikaryon / chemical synaptic transmission / postsynaptic membrane / dendritic spine / postsynaptic density / neuron projection / dendrite / neuronal cell body / glutamatergic synapse / synapse / endoplasmic reticulum membrane / protein kinase binding / cell surface / endoplasmic reticulum / protein-containing complex / membrane / identical protein binding / plasma membrane
Similarity search - Function
PMP-22/EMP/MP20/Claudin family / Voltage-dependent calcium channel, gamma subunit / PMP-22/EMP/MP20/Claudin superfamily / Ionotropic glutamate receptor, metazoa / Ligated ion channel L-glutamate- and glycine-binding site / Ligand-gated ion channel / Ionotropic glutamate receptor, L-glutamate and glycine-binding domain / Ligated ion channel L-glutamate- and glycine-binding site / Ionotropic glutamate receptor / Eukaryotic homologues of bacterial periplasmic substrate binding proteins. ...PMP-22/EMP/MP20/Claudin family / Voltage-dependent calcium channel, gamma subunit / PMP-22/EMP/MP20/Claudin superfamily / Ionotropic glutamate receptor, metazoa / Ligated ion channel L-glutamate- and glycine-binding site / Ligand-gated ion channel / Ionotropic glutamate receptor, L-glutamate and glycine-binding domain / Ligated ion channel L-glutamate- and glycine-binding site / Ionotropic glutamate receptor / Eukaryotic homologues of bacterial periplasmic substrate binding proteins. / Receptor, ligand binding region / Receptor family ligand binding region / Periplasmic binding protein-like I
Similarity search - Domain/homology
CYCLOTHIAZIDE / Glutamate receptor 2 / Voltage-dependent calcium channel gamma-3 subunit
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.02 Å
AuthorsYelshanskaya, M.V. / Sobolevsky, A.I.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS083660 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS107253 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA206573 United States
National Science Foundation (NSF, United States)1818086 United States
CitationJournal: Nature / Year: 2022
Title: Opening of glutamate receptor channel to subconductance levels.
Authors: Maria V Yelshanskaya / Dhilon S Patel / Christopher M Kottke / Maria G Kurnikova / Alexander I Sobolevsky /
Abstract: Ionotropic glutamate receptors (iGluRs) are tetrameric ligand-gated ion channels that open their pores in response to binding of the agonist glutamate. An ionic current through a single iGluR channel ...Ionotropic glutamate receptors (iGluRs) are tetrameric ligand-gated ion channels that open their pores in response to binding of the agonist glutamate. An ionic current through a single iGluR channel shows up to four discrete conductance levels (O1-O4). Higher conductance levels have been associated with an increased number of agonist molecules bound to four individual ligand-binding domains (LBDs). Here we determine structures of a synaptic complex of AMPA-subtype iGluR and the auxiliary subunit γ2 in non-desensitizing conditions with various occupancy of the LBDs by glutamate. We show that glutamate binds to LBDs of subunits B and D only after it is already bound to at least the same number of LBDs that belong to subunits A and C. Our structures combined with single-channel recordings, molecular dynamics simulations and machine-learning analysis suggest that channel opening requires agonist binding to at least two LBDs. Conversely, agonist binding to all four LBDs does not guarantee maximal channel conductance and favours subconductance states O1 and O2, with O3 and O4 being rare and not captured structurally. The lack of subunit independence and low efficiency coupling of glutamate binding to channel opening underlie the gating of synaptic complexes to submaximal conductance levels, which provide a potential for upregulation of synaptic activity.
History
DepositionJan 21, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 20, 2022Provider: repository / Type: Initial release
Revision 1.1May 4, 2022Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2May 18, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Isoform Flip of Glutamate receptor 2,Voltage-dependent calcium channel gamma-3 subunit chimera
B: Isoform Flip of Glutamate receptor 2,Voltage-dependent calcium channel gamma-3 subunit chimera
C: Isoform Flip of Glutamate receptor 2,Voltage-dependent calcium channel gamma-3 subunit chimera
D: Isoform Flip of Glutamate receptor 2,Voltage-dependent calcium channel gamma-3 subunit chimera
hetero molecules


Theoretical massNumber of molelcules
Total (without water)463,3398
Polymers461,7804
Non-polymers1,5604
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Isoform Flip of Glutamate receptor 2,Voltage-dependent calcium channel gamma-3 subunit chimera / GluR-2 / AMPA-selective glutamate receptor 2 / GluR-B / GluR-K2 / Glutamate receptor ionotropic / ...GluR-2 / AMPA-selective glutamate receptor 2 / GluR-B / GluR-K2 / Glutamate receptor ionotropic / AMPA 2 / GluA2 / Neuronal voltage-gated calcium channel gamma-3 subunit / Transmembrane AMPAR regulatory protein gamma-3 / TARP gamma-3


Mass: 115444.922 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gria2, Glur2, Cacng3 / Production host: Homo sapiens (human) / References: UniProt: P19491-2, UniProt: Q8VHX0
#2: Chemical
ChemComp-CYZ / CYCLOTHIAZIDE / 3-BICYCLO[2.2.1]HEPT-5-EN-2-YL-6-CHLORO-3,4- DIHYDRO-2H-1,2,4-BENZOTHIADIAZINE-7-SULFONAMIDE 1,1 DIOXIDE / Cyclothiazide


Mass: 389.878 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C14H16ClN3O4S2 / Feature type: SUBJECT OF INVESTIGATION
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

ComponentName: Ionotropic glutamate receptor GluA2 in complex with auxiliary subunit gamma2 or Stargazin
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 58.5 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 4.02 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 49098 / Symmetry type: POINT

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