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- PDB-5ide: Cryo-EM structure of GluA2/3 AMPA receptor heterotetramer (model I) -

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Basic information

Entry
Database: PDB / ID: 5ide
TitleCryo-EM structure of GluA2/3 AMPA receptor heterotetramer (model I)
Components
  • Glutamate receptor 2GRIA2
  • Glutamate receptor 3
KeywordsSIGNALING PROTEIN / AMPA glutamate receptor
Function / homology
Function and homology information


Trafficking of AMPA receptors / Synaptic adhesion-like molecules / parallel fiber to Purkinje cell synapse / spine synapse / dendritic spine neck / dendritic spine head / Activation of AMPA receptors / response to lithium ion / perisynaptic space / cellular response to glycine ...Trafficking of AMPA receptors / Synaptic adhesion-like molecules / parallel fiber to Purkinje cell synapse / spine synapse / dendritic spine neck / dendritic spine head / Activation of AMPA receptors / response to lithium ion / perisynaptic space / cellular response to glycine / AMPA glutamate receptor activity / Trafficking of GluR2-containing AMPA receptors / immunoglobulin binding / AMPA glutamate receptor complex / kainate selective glutamate receptor activity / ionotropic glutamate receptor complex / extracellularly glutamate-gated ion channel activity / asymmetric synapse / regulation of receptor recycling / Unblocking of NMDA receptors, glutamate binding and activation / glutamate receptor binding / synaptic cleft / positive regulation of synaptic transmission / presynaptic active zone membrane / response to fungicide / glutamate-gated receptor activity / regulation of synaptic transmission, glutamatergic / cellular response to brain-derived neurotrophic factor stimulus / somatodendritic compartment / dendrite membrane / ligand-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / ionotropic glutamate receptor binding / cytoskeletal protein binding / ionotropic glutamate receptor signaling pathway / dendrite cytoplasm / SNARE binding / dendritic shaft / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / synaptic membrane / synaptic transmission, glutamatergic / PDZ domain binding / postsynaptic density membrane / protein tetramerization / modulation of chemical synaptic transmission / Schaffer collateral - CA1 synapse / establishment of protein localization / terminal bouton / receptor internalization / synaptic vesicle membrane / cerebral cortex development / synaptic vesicle / presynapse / signaling receptor activity / presynaptic membrane / amyloid-beta binding / growth cone / chemical synaptic transmission / perikaryon / postsynaptic membrane / scaffold protein binding / dendritic spine / postsynaptic density / neuron projection / axon / dendrite / neuronal cell body / glutamatergic synapse / synapse / protein-containing complex binding / endoplasmic reticulum membrane / protein kinase binding / cell surface / endoplasmic reticulum / protein-containing complex / membrane / identical protein binding / plasma membrane
Similarity search - Function
Bacterial extracellular solute-binding proteins, family 3 / Solute-binding protein family 3/N-terminal domain of MltF / 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. ...Bacterial extracellular solute-binding proteins, family 3 / Solute-binding protein family 3/N-terminal domain of MltF / 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
Glutamate receptor 2 / Glutamate receptor 3
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8.25 Å
AuthorsHerguedas, B. / Garcia-Nafria, J. / Fernandez-Leiro, R. / Greger, I.H.
CitationJournal: Science / Year: 2016
Title: Structure and organization of heteromeric AMPA-type glutamate receptors.
Authors: Beatriz Herguedas / Javier García-Nafría / Ondrej Cais / Rafael Fernández-Leiro / James Krieger / Hinze Ho / Ingo H Greger /
Abstract: AMPA-type glutamate receptors (AMPARs), which are central mediators of rapid neurotransmission and synaptic plasticity, predominantly exist as heteromers of the subunits GluA1 to GluA4. Here we ...AMPA-type glutamate receptors (AMPARs), which are central mediators of rapid neurotransmission and synaptic plasticity, predominantly exist as heteromers of the subunits GluA1 to GluA4. Here we report the first AMPAR heteromer structures, which deviate substantially from existing GluA2 homomer structures. Crystal structures of the GluA2/3 and GluA2/4 N-terminal domains reveal a novel compact conformation with an alternating arrangement of the four subunits around a central axis. This organization is confirmed by cysteine cross-linking in full-length receptors, and it permitted us to determine the structure of an intact GluA2/3 receptor by cryogenic electron microscopy. Two models in the ligand-free state, at resolutions of 8.25 and 10.3 angstroms, exhibit substantial vertical compression and close associations between domain layers, reminiscent of N-methyl-D-aspartate receptors. Model 1 resembles a resting state and model 2 a desensitized state, thus providing snapshots of gating transitions in the nominal absence of ligand. Our data reveal organizational features of heteromeric AMPARs and provide a framework to decipher AMPAR architecture and signaling.
History
DepositionFeb 24, 2016Deposition site: RCSB / Processing site: PDBE
Revision 1.0Mar 16, 2016Provider: repository / Type: Initial release
Revision 1.1Mar 23, 2016Group: Database references
Revision 1.2May 11, 2016Group: Database references
Revision 1.3Jun 15, 2016Group: Database references
Revision 1.4Aug 30, 2017Group: Data collection / Category: em_imaging_optics / em_software
Item: _em_imaging_optics.energyfilter_name / _em_software.name / _em_software.version
Revision 1.5Oct 11, 2017Group: Source and taxonomy / Category: entity_src_gen / Item: _entity_src_gen.host_org_common_name

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Structure visualization

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Assembly

Deposited unit
A: Glutamate receptor 2
B: Glutamate receptor 3
C: Glutamate receptor 2
D: Glutamate receptor 3


Theoretical massNumber of molelcules
Total (without water)393,4784
Polymers393,4784
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Glutamate receptor 2 / GRIA2 / 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: 97663.188 Da / Num. of mol.: 2 / Mutation: N292C
Source method: isolated from a genetically manipulated source
Details: The sequence corresponds to mature rat GluA2 (residues 22-883, isoform Flip, edited at R/G and Q/R sites) with a Myc tag after the first residue and the N292C mutation
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gria2, Glur2 / Plasmid: pires2-EGFP / Cell line (production host): HEK293 gntI- / Production host: Homo sapiens (human) / References: UniProt: P19491
#2: Protein Glutamate receptor 3 / / GluR-3 / AMPA-selective glutamate receptor 3 / GluR-C / GluR-K3 / Glutamate receptor ionotropic / ...GluR-3 / AMPA-selective glutamate receptor 3 / GluR-C / GluR-K3 / Glutamate receptor ionotropic / AMPA 3 / GluA3


Mass: 99075.664 Da / Num. of mol.: 2 / Mutation: R439G, R265C
Source method: isolated from a genetically manipulated source
Details: The sequence corresponds to the mature rat GluA3 subunit (residues 23-888, Flip isoform) with a Flag tag after the first residue and mutated at R439G and R265C
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gria3, Glur3 / Variant: Flip / Plasmid: prk5 / Cell line (production host): HEK293 gntI- / Production host: Homo sapiens (human) / References: UniProt: P19492

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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: AMPA GluA2/3 heterotetramer / Type: COMPLEX
Details: Full-length GluA2/3 heterotetramer containing the A2_N292C and A3_265C mutations
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.4 MDa / Experimental value: NO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293 GntI- / Plasmid: prk5 and pIRES2-EGFP
Buffer solutionpH: 7.4 / Details: 25 mM Tris pH 7.4, 0.25 % DDM, 150 mM NaCl
SpecimenConc.: 0.03 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Incubated for 1 minute, blotted for 3 seconds

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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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 28409 X / Nominal defocus max: 4000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 25 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 980
EM imaging opticsEnergyfilter name: GIF Quantum / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV
Image scansMovie frames/image: 20 / Used frames/image: 1-20

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Processing

EM software
IDNameVersionCategoryDetails
1EMAN2particle selectione2boxer.py was used for initial particle pickingpicking
2RELIONparticle selectionrelion autopick was used for automatic picking
5GctfCTF correction
8UCSF Chimeramodel fitting
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 107939
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 8.25 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 25238 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: For GluA2 chains (A,C), 2 copies of GluA2NTD (3HSY) and two copies of GluA2 LBD (1FTO) were fitted. For GluA3 chains (B,D), 2 copies of GluA3NTD (3O21) and two copies of GluA2 LBD (3UA8) ...Details: For GluA2 chains (A,C), 2 copies of GluA2NTD (3HSY) and two copies of GluA2 LBD (1FTO) were fitted. For GluA3 chains (B,D), 2 copies of GluA3NTD (3O21) and two copies of GluA2 LBD (3UA8) were fitted.For the TMD region, the 4 chains of 3KG2 TMD(residues 509-544 594-629 784-817) were fitted as a rigid body. After fitting the sequence was corrected including mutations and side chains were removed.
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
13HSY

3hsy

B1
21FTO

1fto

B1
33UA8

3ua8

1
43O21

3o21

D1
53KG2

3kg2

1

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