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- PDB-8fpl: LBD conformation 2 (LBDconf2) of GluA2 flip Q isoform of AMPA rec... -

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Basic information

Entry
Database: PDB / ID: 8fpl
TitleLBD conformation 2 (LBDconf2) of GluA2 flip Q isoform of AMPA receptor in complex with gain-of-function TARP gamma-2, with 10mM CaCl2, 150mM NaCl, 1mM MgCl2, 330uM CTZ, and 100uM CNQX (Closed-CaNaMg)
ComponentsGlutamate receptor 2
KeywordsTRANSPORT PROTEIN / ionotropic glutamate receptors / AMPA receptors / ion channel / ligand-gated ion channel / auxiliary subunit / stargazing / TARP gamma2
Function / homology
Function and homology information


spine synapse / dendritic spine neck / dendritic spine head / Activation of AMPA receptors / perisynaptic space / AMPA glutamate receptor activity / ligand-gated monoatomic cation channel activity / Trafficking of GluR2-containing AMPA receptors / response to lithium ion / immunoglobulin binding ...spine synapse / dendritic spine neck / dendritic spine head / Activation of AMPA receptors / perisynaptic space / AMPA glutamate receptor activity / ligand-gated monoatomic cation channel activity / Trafficking of GluR2-containing AMPA receptors / response to lithium ion / immunoglobulin binding / AMPA glutamate receptor complex / kainate selective glutamate receptor activity / ionotropic glutamate receptor complex / extracellularly glutamate-gated ion channel activity / cellular response to glycine / asymmetric synapse / regulation of receptor recycling / Unblocking of NMDA receptors, glutamate binding and activation / positive regulation of synaptic transmission / glutamate receptor binding / extracellular ligand-gated monoatomic ion channel activity / glutamate-gated receptor activity / response to fungicide / glutamate-gated calcium ion channel activity / presynaptic active zone membrane / regulation of synaptic transmission, glutamatergic / somatodendritic compartment / dendrite membrane / cellular response to brain-derived neurotrophic factor stimulus / cytoskeletal protein binding / ligand-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / ionotropic glutamate receptor binding / dendrite cytoplasm / ionotropic glutamate receptor signaling pathway / SNARE binding / dendritic shaft / synaptic transmission, glutamatergic / synaptic membrane / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / PDZ domain binding / protein tetramerization / postsynaptic density membrane / modulation of chemical synaptic transmission / establishment of protein localization / Schaffer collateral - CA1 synapse / terminal bouton / receptor internalization / cerebral cortex development / synaptic vesicle membrane / synaptic vesicle / presynapse / signaling receptor activity / presynaptic membrane / amyloid-beta binding / growth cone / scaffold protein binding / chemical synaptic transmission / perikaryon / postsynaptic membrane / dendritic spine / postsynaptic density / neuron projection / axon / neuronal cell body / glutamatergic synapse / dendrite / synapse / protein-containing complex binding / protein kinase binding / cell surface / endoplasmic reticulum / protein-containing complex / identical protein binding / membrane / plasma membrane
Similarity search - Function
Ionotropic glutamate receptor, metazoa / Ligated ion channel L-glutamate- and glycine-binding site / Ionotropic glutamate receptor, L-glutamate and glycine-binding domain / Ligated ion channel L-glutamate- and glycine-binding site / Ligand-gated ion channel / : / 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
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.81 Å
AuthorsNakagawa, T.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)MH123474 United States
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: The open gate of the AMPA receptor forms a Ca binding site critical in regulating ion transport.
Authors: Terunaga Nakagawa / Xin-Tong Wang / Federico J Miguez-Cabello / Derek Bowie /
Abstract: Alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid receptors (AMPARs) are cation-selective ion channels that mediate most fast excitatory neurotransmission in the brain. Although their gating ...Alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid receptors (AMPARs) are cation-selective ion channels that mediate most fast excitatory neurotransmission in the brain. Although their gating mechanism has been studied extensively, understanding how cations traverse the pore has remained elusive. Here we investigated putative ion and water densities in the open pore of Ca-permeable AMPARs (rat GRIA2 flip-Q isoform) at 2.3-2.6 Å resolution. We show that the ion permeation pathway attains an extracellular Ca binding site (site-G) when the channel gate moves into the open configuration. Site-G is highly selective for Ca over Na, favoring the movement of Ca into the selectivity filter of the pore. Seizure-related N619K mutation, adjacent to site-G, promotes channel opening but attenuates Ca binding and thus diminishes Ca permeability. Our work identifies the importance of site-G, which coordinates with the Q/R site of the selectivity filter to ensure the preferential transport of Ca through the channel pore.
History
DepositionJan 4, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 28, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Database references / Category: citation_author / Item: _citation_author.name
Revision 1.2Mar 13, 2024Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3May 1, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.4Nov 20, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Glutamate receptor 2
C: Glutamate receptor 2
A: Glutamate receptor 2
D: Glutamate receptor 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)400,74212
Polymers398,2384
Non-polymers2,5048
Water00
1


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

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Components

#1: Protein
Glutamate receptor 2 / 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


Mass: 99559.461 Da / Num. of mol.: 4 / Mutation: FLAG epitope tag (DYKDDDDK) insertion
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gria2, Glur2 / Cell line (production host): HEK / Production host: Homo sapiens (human) / References: UniProt: P19491
#2: Chemical
ChemComp-Y4Y / 6-(aminomethyl)-7-nitro-1,4-dihydroquinoxaline-2,3-dione


Mass: 236.184 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C9H8N4O4 / Feature type: SUBJECT OF INVESTIGATION
#3: 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


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

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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: GluA2 flip-Q isoform in complex with TARP gamma2 (K52E, K53E)
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.5 MDa / Experimental value: NO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK
Buffer solutionpH: 8
Details: 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, final conc. 0.1mM) and cyclothiazide (CTZ, final conc. 0.33mM) was added before freezing.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris1
2150 mMSodium ChlorideNaCl1
310 mMCalcium ChlorideCaCl21
41 mMMagnesium ChlorideMgCl21
50.03 %GDN1
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 56 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 34553

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Processing

EM software
IDNameCategory
1RELIONparticle selection
2EPUimage acquisition
4CTFFINDCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.81 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 262250 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0038348
ELECTRON MICROSCOPYf_angle_d0.46811280
ELECTRON MICROSCOPYf_dihedral_angle_d4.0161200
ELECTRON MICROSCOPYf_chiral_restr0.0411240
ELECTRON MICROSCOPYf_plane_restr0.0031386

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