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- PDB-5kbt: Cryo-EM structure of GluA2-1xSTZ complex at 6.4 Angstrom resolution -

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Basic information

Entry
Database: PDB / ID: 5kbt
TitleCryo-EM structure of GluA2-1xSTZ complex at 6.4 Angstrom resolution
ComponentsGlutamate receptor 2,Voltage-dependent calcium channel gamma-2 subunit
KeywordsTRANSPORT PROTEIN / Cryo-EM
Function / homologyVoltage-dependent calcium channel, gamma subunit / Trafficking of AMPA receptors / Ligand-gated ion channel / PMP-22/EMP/MP20/Claudin family / Ionotropic glutamate receptor, L-glutamate and glycine-binding domain / Receptor family ligand binding region / Ligated ion channel L-glutamate- and glycine-binding site / Periplasmic binding protein-like I / Presynaptic depolarization and calcium channel opening / Ionotropic glutamate receptor, metazoa ...Voltage-dependent calcium channel, gamma subunit / Trafficking of AMPA receptors / Ligand-gated ion channel / PMP-22/EMP/MP20/Claudin family / Ionotropic glutamate receptor, L-glutamate and glycine-binding domain / Receptor family ligand binding region / Ligated ion channel L-glutamate- and glycine-binding site / Periplasmic binding protein-like I / Presynaptic depolarization and calcium channel opening / Ionotropic glutamate receptor, metazoa / Ionotropic glutamate receptor / Receptor, ligand binding region / Phase 2 - plateau phase / PMP-22/EMP/MP20/Claudin superfamily / Voltage-dependent calcium channel, gamma-2 subunit / LGI-ADAM interactions / Phase 0 - rapid depolarisation / regulation of postsynaptic neurotransmitter receptor activity / neurotransmitter receptor transport, postsynaptic endosome to lysosome / channel regulator activity / neurotransmitter receptor internalization / postsynaptic density membrane / nervous system process / regulation of AMPA receptor activity / postsynaptic neurotransmitter receptor diffusion trapping / voltage-gated calcium channel complex / somatodendritic compartment / neuromuscular junction development / membrane hyperpolarization / transmission of nerve impulse / membrane depolarization / voltage-gated calcium channel activity / asymmetric synapse / AMPA glutamate receptor activity / regulation of membrane potential / AMPA glutamate receptor complex / kainate selective glutamate receptor activity / ionotropic glutamate receptor complex / response to lithium ion / regulation of synaptic transmission, glutamatergic / regulation of receptor recycling / positive regulation of synaptic transmission / SNARE binding / response to fungicide / synaptic vesicle membrane / ionotropic glutamate receptor activity / extracellularly glutamate-gated ion channel activity / ionotropic glutamate receptor signaling pathway / dendritic shaft / PDZ domain binding / presynaptic membrane / dendrite cytoplasm / receptor internalization / terminal bouton / chemical synaptic transmission / growth cone / establishment of protein localization / signaling receptor activity / protein tetramerization / amyloid-beta binding / postsynaptic membrane / ATPase binding / perikaryon / postsynaptic density / neuron projection / dendritic spine / synapse / cell junction / neuronal cell body / dendrite / intracellular / endoplasmic reticulum membrane / integral component of plasma membrane / cell surface / protein-containing complex / identical protein binding / Voltage-dependent calcium channel gamma-2 subunit / Glutamate receptor 2
Function and homology information
Specimen sourceRattus norvegicus (Norway rat)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 6.4 Å resolution
AuthorsTwomey, E.C. / Yelshanskaya, M.V. / Grassucci, R.A. / 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 typeGroupCategoryProviderType
1.0Jul 13, 2016Structure modelrepositoryInitial release
1.1Jul 20, 2016Structure modelOther
1.2Sep 27, 2017Structure modelAuthor supporting evidencepdbx_audit_support

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)464,58612
Polyers462,0644
Non-polymers2,5228
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 115515.984 Da / Num. of mol.: 4 / Mutation: N241E, V382L, G384E, N385D, V758L
Source: (gene. exp.) Rattus norvegicus (Norway rat), (gene. exp.) Mus musculus (house mouse)
Gene: Gria2, Glur2, Cacng2, Stg / Production host: Homo sapiens (human) / References: UniProt: P19491, UniProt: O88602
#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, 3 / Source: RECOMBINANT
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Cell: HEK293 / Organism: Homo sapiens (human) / Plasmid: Bacmam
Buffer solutionpH: 8
SpecimenConc.: 2 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: 295 kelvins / Details: 3 blot force, 8.0 s blot time

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 80 e/Å2
Details: 40 frames were collected across 8 seconds per image.
Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 1
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategory
1SerialEMimage acquisition
3CTFFIND4.0CTF correction
9RELION1.4initial Euler assignment
10RELION1.4final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1
3D reconstructionResolution: 6.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 39972 / Symmetry type: POINT

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