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- PDB-5weo: Activated GluA2 complex bound to glutamate, cyclothiazide, and ST... -

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Basic information

Entry
Database: PDB / ID: 5weo
TitleActivated GluA2 complex bound to glutamate, cyclothiazide, and STZ in digitonin
ComponentsGlutamate receptor 2,Voltage-dependent calcium channel gamma-2 subunit chimera
KeywordsTRANSPORT PROTEIN / Ion channel
Function / homology
Function and homology information


Presynaptic depolarization and calcium channel opening / LGI-ADAM interactions / Trafficking of AMPA receptors / eye blink reflex / positive regulation of protein localization to basolateral plasma membrane / positive regulation of AMPA receptor activity / cerebellar mossy fiber / postsynaptic neurotransmitter receptor diffusion trapping / membrane hyperpolarization / regulation of AMPA receptor activity ...Presynaptic depolarization and calcium channel opening / LGI-ADAM interactions / Trafficking of AMPA receptors / eye blink reflex / positive regulation of protein localization to basolateral plasma membrane / positive regulation of AMPA receptor activity / cerebellar mossy fiber / postsynaptic neurotransmitter receptor diffusion trapping / membrane hyperpolarization / regulation of AMPA receptor activity / channel regulator activity / protein targeting to membrane / nervous system process / voltage-gated calcium channel complex / regulation of synaptic plasticity by chemical substance / spine synapse / dendritic spine neck / dendritic spine cytoplasm / dendritic spine head / cellular response to amine stimulus / Activation of AMPA receptors / neurotransmitter receptor localization to postsynaptic specialization membrane / ligand-gated monoatomic cation channel activity / perisynaptic space / Trafficking of GluR2-containing AMPA receptors / response to lithium ion / AMPA glutamate receptor activity / AMPA glutamate receptor clustering / neuromuscular junction development / transmission of nerve impulse / regulation of receptor recycling / kainate selective glutamate receptor activity / immunoglobulin binding / AMPA glutamate receptor complex / extracellularly glutamate-gated ion channel activity / ionotropic glutamate receptor complex / cellular response to glycine / membrane depolarization / asymmetric synapse / Unblocking of NMDA receptors, glutamate binding and activation / glutamate receptor binding / positive regulation of synaptic transmission / regulation of postsynaptic membrane neurotransmitter receptor levels / conditioned place preference / voltage-gated calcium channel activity / response to fungicide / regulation of synaptic transmission, glutamatergic / extracellular ligand-gated monoatomic ion channel activity / cytoskeletal protein binding / glutamate-gated receptor activity / cellular response to brain-derived neurotrophic factor stimulus / regulation of long-term synaptic depression / positive regulation of synaptic transmission, glutamatergic / glutamate-gated calcium ion channel activity / somatodendritic compartment / presynaptic active zone membrane / ionotropic glutamate receptor binding / ionotropic glutamate receptor signaling pathway / dendrite cytoplasm / dendrite membrane / excitatory synapse / ligand-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / positive regulation of excitatory postsynaptic potential / hippocampal mossy fiber to CA3 synapse / dendritic shaft / SNARE binding / synaptic membrane / PDZ domain binding / establishment of protein localization / protein tetramerization / synaptic transmission, glutamatergic / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / receptor internalization / cerebral cortex development / regulation of membrane potential / response to calcium ion / postsynaptic density membrane / modulation of chemical synaptic transmission / long-term synaptic potentiation / Schaffer collateral - CA1 synapse / terminal bouton / synaptic vesicle / synaptic vesicle membrane / amyloid-beta binding / presynapse / growth cone / signaling receptor activity / presynaptic membrane / scaffold protein binding / chemical synaptic transmission / dendritic spine / perikaryon / postsynaptic membrane / postsynaptic density / neuron projection / external side of plasma membrane / axon / neuronal cell body / synapse / dendrite
Similarity search - Function
Voltage-dependent calcium channel, gamma-2 subunit / : / PMP-22/EMP/MP20/Claudin family / Voltage-dependent calcium channel, gamma subunit / PMP-22/EMP/MP20/Claudin superfamily / Ionotropic glutamate receptor, metazoa / Ligated ion channel L-glutamate- and glycine-binding site / Ligand-gated ion channel / Ionotropic glutamate receptor, L-glutamate and glycine-binding domain / Ligated ion channel L-glutamate- and glycine-binding site ...Voltage-dependent calcium channel, gamma-2 subunit / : / PMP-22/EMP/MP20/Claudin family / Voltage-dependent calcium channel, gamma subunit / PMP-22/EMP/MP20/Claudin superfamily / Ionotropic glutamate receptor, metazoa / Ligated ion channel L-glutamate- and glycine-binding site / Ligand-gated ion channel / Ionotropic glutamate receptor, L-glutamate and glycine-binding domain / Ligated ion channel L-glutamate- and glycine-binding site / : / 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 / GLUTAMIC ACID / Voltage-dependent calcium channel gamma-2 subunit / Glutamate receptor 2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsTwomey, E.C. / Yelshanskaya, M.V. / Grassucci, R.A. / Frank, J. / Sobolevsky, A.I.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS093838 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS083660 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA206573 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM029169 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nature / Year: 2017
Title: Channel opening and gating mechanism in AMPA-subtype glutamate receptors.
Authors: Edward C Twomey / Maria V Yelshanskaya / Robert A Grassucci / Joachim Frank / Alexander I Sobolevsky /
Abstract: AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-subtype ionotropic glutamate receptors mediate fast excitatory neurotransmission throughout the central nervous system. Gated by the ...AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-subtype ionotropic glutamate receptors mediate fast excitatory neurotransmission throughout the central nervous system. Gated by the neurotransmitter glutamate, AMPA receptors are critical for synaptic strength, and dysregulation of AMPA receptor-mediated signalling is linked to numerous neurological diseases. Here we use cryo-electron microscopy to solve the structures of AMPA receptor-auxiliary subunit complexes in the apo, antagonist- and agonist-bound states and determine the iris-like mechanism of ion channel opening. The ion channel selectivity filter is formed by the extended portions of the re-entrant M2 loops, while the helical portions of M2 contribute to extensive hydrophobic interfaces between AMPA receptor subunits in the ion channel. We show how the permeation pathway changes upon channel opening and identify conformational changes throughout the entire AMPA receptor that accompany activation and desensitization. Our findings provide a framework for understanding gating across the family of ionotropic glutamate receptors and the role of AMPA receptors in excitatory neurotransmission.
History
DepositionJul 10, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 2, 2017Provider: repository / Type: Initial release
Revision 1.1Sep 13, 2017Group: Author supporting evidence / Database references / Category: citation / pdbx_audit_support
Item: _citation.journal_id_ISSN / _citation.journal_volume ..._citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _pdbx_audit_support.funding_organization
Revision 1.2Jul 18, 2018Group: Data collection / Experimental preparation / Category: em_sample_support / Item: _em_sample_support.grid_type
Revision 1.3Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Oct 23, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _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,Voltage-dependent calcium channel gamma-2 subunit chimera
B: Glutamate receptor 2,Voltage-dependent calcium channel gamma-2 subunit chimera
C: Glutamate receptor 2,Voltage-dependent calcium channel gamma-2 subunit chimera
D: Glutamate receptor 2,Voltage-dependent calcium channel gamma-2 subunit chimera
hetero molecules


Theoretical massNumber of molelcules
Total (without water)464,15612
Polymers462,0084
Non-polymers2,1488
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area30380 Å2
ΔGint-237 kcal/mol
Surface area191300 Å2

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Components

#1: Protein
Glutamate receptor 2,Voltage-dependent calcium channel gamma-2 subunit chimera / 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: 115501.969 Da / Num. of mol.: 4
Fragment: UNP P19491 residues 25-847, UNP O88602 2-208 linked via LINKER GT
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 / Cell line (production host): HEK293 gnti- / Production host: Homo sapiens (human) / References: UniProt: P19491, UniProt: O88602
#2: Chemical
ChemComp-GLU / GLUTAMIC ACID


Type: L-peptide linking / Mass: 147.129 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C5H9NO4
#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
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: Activated GluA2 complex bound to glutamate, cyclothiazide, and STZ in digitonin
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293 gnti-
Buffer solutionpH: 8
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Activated GluA2 complex bound to glutamate, cyclothiazide, and STZ in digitonin
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K

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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 FIELD
Image recordingAverage exposure time: 8 sec. / Electron dose: 55 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 69207
Details: TMD map uploaded under additional files is at 4.0 angstrom resolution.
Symmetry type: POINT
Atomic model buildingSpace: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00831856
ELECTRON MICROSCOPYf_angle_d0.97743066
ELECTRON MICROSCOPYf_dihedral_angle_d10.88718730
ELECTRON MICROSCOPYf_chiral_restr0.0554774
ELECTRON MICROSCOPYf_plane_restr0.0075408

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