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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 2GRIA2
KeywordsTRANSPORT PROTEIN / Cryo-EM
Function / homologyIonotropic glutamate receptor / Ionotropic glutamate receptor, metazoa / Ligated ion channel L-glutamate- and glycine-binding site / Receptor family ligand binding region / Ligand-gated ion channel / Periplasmic binding protein-like I / Ionotropic glutamate receptor, L-glutamate and glycine-binding domain / Receptor, ligand binding region / AMPA glutamate receptor activity / asymmetric synapse ...Ionotropic glutamate receptor / Ionotropic glutamate receptor, metazoa / Ligated ion channel L-glutamate- and glycine-binding site / Receptor family ligand binding region / Ligand-gated ion channel / Periplasmic binding protein-like I / Ionotropic glutamate receptor, L-glutamate and glycine-binding domain / Receptor, ligand binding region / AMPA glutamate receptor activity / asymmetric synapse / AMPA glutamate receptor complex / kainate selective glutamate receptor activity / ionotropic glutamate receptor complex / response to lithium ion / regulation of receptor recycling / SNARE binding / transmitter-gated ion channel activity involved in regulation of postsynaptic membrane potential / positive regulation of synaptic transmission / integral component of postsynaptic membrane / regulation of synaptic transmission, glutamatergic / response to fungicide / integral component of postsynaptic density membrane / ionotropic glutamate receptor activity / ionotropic glutamate receptor signaling pathway / cytoskeletal protein binding / synaptic vesicle membrane / dendritic shaft / dendrite cytoplasm / receptor internalization / integral component of presynaptic membrane / PDZ domain binding / Schaffer collateral - CA1 synapse / presynaptic membrane / terminal bouton / establishment of protein localization / protein tetramerization / growth cone / chemical synaptic transmission / amyloid-beta binding / ATPase binding / perikaryon / signaling receptor activity / dendritic spine / postsynaptic density / cell junction / synapse / glutamatergic synapse / dendrite / endoplasmic reticulum membrane / neuronal cell body / protein kinase binding / integral component of plasma membrane / cell surface / protein-containing complex / identical protein binding / plasma membrane / Glutamate receptor 2
Function and homology information
Specimen sourceRattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 6.8 Å resolution
AuthorsTwomey, E.C. / Yelshanskaya, M.V. / Grassucci, R.G. / Frank, J. / Sobolevsky, A.I.
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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jun 3, 2016 / Release: Jul 13, 2016
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jul 13, 2016Structure modelrepositoryInitial release
1.1Jul 20, 2016Structure modelOther
1.2Sep 27, 2017Structure modelAuthor supporting evidencepdbx_audit_support_pdbx_audit_support.funding_organization

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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
Polyers369,0094
Non-polymers2,5228
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein/peptide
Glutamate receptor 2 / GRIA2 / 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: (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 / Formula: C14H15F3N3O6P
#3: Chemical
ChemComp-NAG / N-ACETYL-D-GLUCOSAMINE


Mass: 221.208 Da / Num. of mol.: 4 / Formula: C8H15NO6 / N-Acetylglucosamine

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 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)Cell: HEK293 / Organism: Homo sapiens (human) / 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 / Grid type: C-flat Au 1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 299 kelvins / 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
MicroscopyMicroscope model: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Alignment procedure: COMA FREE
Image recordingAverage exposure time: 9 sec. / Electron dose: 45 e/Å2
Details: 30 frames across 9 seconds were collected per image.
Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 1
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
3D reconstructionResolution: 6.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 33152 / Symmetry type: POINT

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