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- PDB-5kbu: Cryo-EM structure of GluA2-2xSTZ complex at 7.8 Angstrom resolution -

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Basic information

Entry
Database: PDB / ID: 5kbu
TitleCryo-EM structure of GluA2-2xSTZ complex at 7.8 Angstrom resolution
ComponentsGlutamate receptor 2,Voltage-dependent calcium channel gamma-2 subunit
KeywordsTRANSPORT PROTEIN / Cryo-EM
Function / homology
Function and homology information


positive regulation of protein localization to basolateral plasma membrane / eye blink reflex / neurotransmitter receptor transport, postsynaptic endosome to lysosome / regulation of postsynaptic membrane neurotransmitter receptor levels / regulation of postsynaptic neurotransmitter receptor activity / neurotransmitter receptor internalization / positive regulation of AMPA receptor activity / regulation of AMPA receptor activity / cerebellar mossy fiber / neurotransmitter receptor localization to postsynaptic specialization membrane ...positive regulation of protein localization to basolateral plasma membrane / eye blink reflex / neurotransmitter receptor transport, postsynaptic endosome to lysosome / regulation of postsynaptic membrane neurotransmitter receptor levels / regulation of postsynaptic neurotransmitter receptor activity / neurotransmitter receptor internalization / positive regulation of AMPA receptor activity / regulation of AMPA receptor activity / cerebellar mossy fiber / neurotransmitter receptor localization to postsynaptic specialization membrane / postsynaptic neurotransmitter receptor diffusion trapping / membrane hyperpolarization / protein targeting to membrane / voltage-gated calcium channel complex / neuromuscular junction development / transmission of nerve impulse / membrane depolarization / nervous system process / AMPA glutamate receptor activity / voltage-gated calcium channel activity / asymmetric synapse / ionotropic glutamate receptor binding / extracellularly glutamate-gated ion channel activity / kainate selective glutamate receptor activity / AMPA glutamate receptor complex / ionotropic glutamate receptor complex / response to lithium ion / immunoglobulin binding / regulation of receptor recycling / somatodendritic compartment / positive regulation of synaptic transmission / integral component of postsynaptic membrane / SNARE binding / ionotropic glutamate receptor activity / response to fungicide / regulation of synaptic transmission, glutamatergic / integral component of postsynaptic density membrane / synaptic membrane / glutamate receptor activity / transmitter-gated ion channel activity involved in regulation of postsynaptic membrane potential / synaptic vesicle membrane / ionotropic glutamate receptor signaling pathway / synaptic transmission, glutamatergic / cytoskeletal protein binding / dendritic shaft / regulation of membrane potential / hippocampal mossy fiber to CA3 synapse / postsynaptic density membrane / dendrite cytoplasm / integral component of presynaptic membrane / PDZ domain binding / receptor internalization / Schaffer collateral - CA1 synapse / presynaptic membrane / protein tetramerization / terminal bouton / response to calcium ion / establishment of protein localization / modulation of chemical synaptic transmission / growth cone / postsynaptic membrane / chemical synaptic transmission / synaptic vesicle / amyloid-beta binding / ATPase binding / perikaryon / signaling receptor activity / dendritic spine / postsynaptic density / neuron projection / synapse / cell junction / glutamatergic synapse / dendrite / endoplasmic reticulum membrane / neuronal cell body / protein kinase binding / endoplasmic reticulum / integral component of plasma membrane / cell surface / protein-containing complex / membrane / identical protein binding / plasma membrane / cytosol
Ligated ion channel L-glutamate- and glycine-binding site / Ionotropic glutamate receptor, metazoa / PMP-22/EMP/MP20/Claudin superfamily / Ionotropic glutamate receptor / Voltage-dependent calcium channel, gamma-2 subunit / Voltage-dependent calcium channel, gamma subunit / Ionotropic glutamate receptor, L-glutamate and glycine-binding domain / Periplasmic binding protein-like I / PMP-22/EMP/MP20/Claudin family / Ligand-gated ion channel ...Ligated ion channel L-glutamate- and glycine-binding site / Ionotropic glutamate receptor, metazoa / PMP-22/EMP/MP20/Claudin superfamily / Ionotropic glutamate receptor / Voltage-dependent calcium channel, gamma-2 subunit / Voltage-dependent calcium channel, gamma subunit / Ionotropic glutamate receptor, L-glutamate and glycine-binding domain / Periplasmic binding protein-like I / PMP-22/EMP/MP20/Claudin family / Ligand-gated ion channel / Receptor family ligand binding region / Receptor, ligand binding region
Voltage-dependent calcium channel gamma-2 subunit / Glutamate receptor 2
Biological speciesRattus norvegicus (Norway rat)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.8 Å
AuthorsTwomey, E.C. / Yelshanskaya, M.V. / Grassucci, R.A. / 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.
Validation Report
SummaryFull reportAbout validation report
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

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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
Polymers462,0644
Non-polymers2,5228
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Glutamate receptor 2,Voltage-dependent calcium channel gamma-2 subunit / 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-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 method: isolated from a genetically manipulated source
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 / Fanapanel


Mass: 409.254 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C14H15F3N3O6P / Comment: antagonist, medication*YM
#3: Sugar
ChemComp-NAG / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Mass: 221.208 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
Compound detailsThe protein was expressed as a tandem fusion construct (GluA2 fused to stargazin, also named TARP- ...The protein was expressed as a tandem fusion construct (GluA2 fused to stargazin, also named TARP-y2 or cagcn2). However, this second protein which is covalently linked or fused to the C-terminus of GluA2 is not observed in chains B and D in this structure.

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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, 3 / Source: RECOMBINANT
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293 / 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 divisions/in. / Grid type: C-flat Au 1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K / 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
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy
Specimen holderModel: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 80 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1
Details: 40 frames were collected across 8 seconds per image.
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategory
1SerialEMimage acquisition
3CTFFIND4CTF correction
9RELION1.4initial Euler assignment
10RELION1.4final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 7.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 10293 / Symmetry type: POINT

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