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- PDB-5kbv: Cryo-EM structure of GluA2 bound to antagonist ZK200775 at 6.8 An... -

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Basic information

Entry
Database: PDB / ID: 5kbv
TitleCryo-EM structure of GluA2 bound to antagonist ZK200775 at 6.8 Angstrom resolution
ComponentsGlutamate receptor 2
KeywordsTRANSPORT PROTEIN / Cryo-EM
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 / cellular response to glycine / extracellularly glutamate-gated ion channel activity / asymmetric synapse / regulation of receptor recycling / Unblocking of NMDA receptors, glutamate binding and activation / positive regulation of synaptic transmission / glutamate receptor binding / glutamate-gated receptor activity / regulation of synaptic transmission, glutamatergic / response to fungicide / cytoskeletal protein binding / ionotropic glutamate receptor binding / presynaptic active zone membrane / extracellular ligand-gated monoatomic ion channel activity / somatodendritic compartment / glutamate-gated calcium ion channel activity / cellular response to brain-derived neurotrophic factor stimulus / ligand-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / dendrite membrane / dendrite cytoplasm / ionotropic glutamate receptor signaling pathway / SNARE binding / dendritic shaft / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / synaptic transmission, glutamatergic / PDZ domain binding / protein tetramerization / establishment of protein localization / synaptic membrane / modulation of chemical synaptic transmission / postsynaptic density membrane / terminal bouton / Schaffer collateral - CA1 synapse / cerebral cortex development / receptor internalization / synaptic vesicle membrane / synaptic vesicle / presynapse / signaling receptor activity / amyloid-beta binding / presynaptic membrane / growth cone / scaffold protein binding / chemical synaptic transmission / dendritic spine / perikaryon / postsynaptic membrane / neuron projection / postsynaptic density / axon / neuronal cell body / synapse / dendrite / protein-containing complex binding / protein kinase binding / glutamatergic synapse / 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
Chem-ZK1 / Glutamate receptor 2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.8 Å
AuthorsTwomey, E.C. / Yelshanskaya, M.V. / Grassucci, R.G. / Frank, J. / Sobolevsky, A.I.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32 GM008224, T32 GM008281, R01 NS083660, R01 GM29169 United States
CitationJournal: Science / Year: 2016
Title: Elucidation of AMPA receptor-stargazin complexes by cryo-electron microscopy.
Authors: Edward C Twomey / Maria V Yelshanskaya / Robert A Grassucci / Joachim Frank / Alexander I Sobolevsky /
Abstract: AMPA-subtype ionotropic glutamate receptors (AMPARs) mediate fast excitatory neurotransmission and contribute to high cognitive processes such as learning and memory. In the brain, AMPAR trafficking, ...AMPA-subtype ionotropic glutamate receptors (AMPARs) mediate fast excitatory neurotransmission and contribute to high cognitive processes such as learning and memory. In the brain, AMPAR trafficking, gating, and pharmacology is tightly controlled by transmembrane AMPAR regulatory proteins (TARPs). Here, we used cryo-electron microscopy to elucidate the structural basis of AMPAR regulation by one of these auxiliary proteins, TARP γ2, or stargazin (STZ). Our structures illuminate the variable interaction stoichiometry of the AMPAR-TARP complex, with one or two TARP molecules binding one tetrameric AMPAR. Analysis of the AMPAR-STZ binding interfaces suggests that electrostatic interactions between the extracellular domains of AMPAR and STZ play an important role in modulating AMPAR function through contact surfaces that are conserved across AMPARs and TARPs. We propose a model explaining how TARPs stabilize the activated state of AMPARs and how the interactions between AMPARs and their auxiliary proteins control fast excitatory synaptic transmission.
History
DepositionJun 3, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 13, 2016Provider: repository / Type: Initial release
Revision 1.1Jul 20, 2016Group: Other
Revision 1.2Sep 27, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Dec 25, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Jul 29, 2020Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly
Item: _chem_comp.name / _chem_comp.type ..._chem_comp.name / _chem_comp.type / _entity.pdbx_description / _pdbx_entity_nonpoly.name
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 1.5Nov 20, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI ..._chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)371,53112
Polymers369,0094
Non-polymers2,5228
Water00
1


  • Idetical with deposited unit
  • defined by author
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: 92252.344 Da / Num. of mol.: 4 / Mutation: N241E, V382L,G384E, N385D, V758L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gria2, Glur2 / Production host: Homo sapiens (human) / References: UniProt: P19491
#2: Chemical
ChemComp-ZK1 / {[7-morpholin-4-yl-2,3-dioxo-6-(trifluoromethyl)-3,4-dihydroquinoxalin-1(2H)-yl]methyl}phosphonic acid / [[3,4-Dihydro-7-(4-morpholinyl)-2,3-dioxo-6-(trifluorom ethyl)-1(2H)-quinoxalinyl]methyl]phosphonic acid


Mass: 409.254 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C14H15F3N3O6P / Comment: antagonist, medication*YM
#3: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 4
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
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: Protein / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293 / Plasmid: Bacmam
Buffer solutionpH: 8
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Grid coated with gold prior to use. / Grid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: C-flat Au 1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 299 K / Details: Blot force 3, 8.0 s blot time

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Alignment procedure: COMA FREE
Image recordingAverage exposure time: 9 sec. / Electron dose: 45 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1
Details: 30 frames across 9 seconds were collected per image.
Image scansMovie frames/image: 30 / Used frames/image: 1-30

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Processing

EM software
IDNameVersionCategory
1Leginonimage acquisition
3CTFFIND4CTF correction
9RELION1.4initial Euler assignment
10RELION1.4final Euler assignment
11RELION1.43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 6.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 33152 / Symmetry type: POINT

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