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- EMDB-43276: GluA2 bound to GYKI-52466 and Glutamate, Inhibited State 2 -

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Basic information

Entry
Database: EMDB / ID: EMD-43276
TitleGluA2 bound to GYKI-52466 and Glutamate, Inhibited State 2
Map dataInhibited state 2 full map
Sample
  • Complex: AMPA Receptor GluA2 Homotetramer
    • Protein or peptide: Isoform Flip of Glutamate receptor 2
  • Ligand: GLUTAMIC ACID
  • Ligand: 4-[(5S,8R)-8-methyl-6,7,8,9-tetrahydro-2H,5H-[1,3]dioxolo[4,5-h][2,3]benzodiazepin-5-yl]aniline
Keywordsligand gated ion channel / ionotropic glutamate receptor / allosteric inhibition / MEMBRANE PROTEIN / MEMBRANE PROTEIN-INHIBITOR complex
Function / homology
Function and homology information


spine synapse / dendritic spine neck / dendritic spine head / Activation of AMPA receptors / perisynaptic space / AMPA glutamate receptor activity / ligand-gated monoatomic cation channel activity / Trafficking of GluR2-containing AMPA receptors / response to lithium ion / immunoglobulin binding ...spine synapse / dendritic spine neck / dendritic spine head / Activation of AMPA receptors / perisynaptic space / AMPA glutamate receptor activity / ligand-gated monoatomic cation channel activity / Trafficking of GluR2-containing AMPA receptors / response to lithium ion / immunoglobulin binding / AMPA glutamate receptor complex / kainate selective glutamate receptor activity / ionotropic glutamate receptor complex / cellular response to glycine / extracellularly glutamate-gated ion channel activity / asymmetric synapse / regulation of receptor recycling / Unblocking of NMDA receptors, glutamate binding and activation / positive regulation of synaptic transmission / glutamate receptor binding / glutamate-gated receptor activity / regulation of synaptic transmission, glutamatergic / response to fungicide / cytoskeletal protein binding / ionotropic glutamate receptor binding / presynaptic active zone membrane / extracellular ligand-gated monoatomic ion channel activity / somatodendritic compartment / glutamate-gated calcium ion channel activity / cellular response to brain-derived neurotrophic factor stimulus / ligand-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / dendrite membrane / dendrite cytoplasm / ionotropic glutamate receptor signaling pathway / SNARE binding / dendritic shaft / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / synaptic transmission, glutamatergic / PDZ domain binding / protein tetramerization / establishment of protein localization / synaptic membrane / modulation of chemical synaptic transmission / postsynaptic density membrane / terminal bouton / Schaffer collateral - CA1 synapse / cerebral cortex development / receptor internalization / synaptic vesicle membrane / synaptic vesicle / presynapse / signaling receptor activity / amyloid-beta binding / presynaptic membrane / growth cone / scaffold protein binding / chemical synaptic transmission / dendritic spine / perikaryon / postsynaptic membrane / neuron projection / postsynaptic density / axon / neuronal cell body / synapse / dendrite / protein-containing complex binding / protein kinase binding / glutamatergic synapse / cell surface / endoplasmic reticulum / protein-containing complex / identical protein binding / membrane / plasma membrane
Similarity search - Function
Ionotropic glutamate receptor, metazoa / Ligated ion channel L-glutamate- and glycine-binding site / Ionotropic glutamate receptor, L-glutamate and glycine-binding domain / Ligated ion channel L-glutamate- and glycine-binding site / Ligand-gated ion channel / : / 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
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.85 Å
AuthorsHale WD / Montano Romero A / Huganir RL / Twomey EC
Funding support United States, 5 items
OrganizationGrant numberCountry
Other privateKinship Foundation 22098168
Other privateDiana Helis Henry Medical Research Foundation 142548
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)MH112152 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS036715 United States
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)MH132811 United States
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Allosteric competition and inhibition in AMPA receptors.
Authors: W Dylan Hale / Alejandra Montaño Romero / Cuauhtemoc U Gonzalez / Vasanthi Jayaraman / Albert Y Lau / Richard L Huganir / Edward C Twomey /
Abstract: Excitatory neurotransmission is principally mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-subtype ionotropic glutamate receptors (AMPARs). Negative allosteric modulators ...Excitatory neurotransmission is principally mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-subtype ionotropic glutamate receptors (AMPARs). Negative allosteric modulators are therapeutic candidates that inhibit AMPAR activation and can compete with positive modulators to control AMPAR function through unresolved mechanisms. Here we show that allosteric inhibition pushes AMPARs into a distinct state that prevents both activation and positive allosteric modulation. We used cryo-electron microscopy to capture AMPARs bound to glutamate, while a negative allosteric modulator, GYKI-52466, and positive allosteric modulator, cyclothiazide, compete for control of the AMPARs. GYKI-52466 binds in the ion channel collar and inhibits AMPARs by decoupling the ligand-binding domains from the ion channel. The rearrangement of the ligand-binding domains ruptures the cyclothiazide site, preventing positive modulation. Our data provide a framework for understanding allostery of AMPARs and for rational design of therapeutics targeting AMPARs in neurological diseases.
History
DepositionJan 5, 2024-
Header (metadata) releaseJun 5, 2024-
Map releaseJun 5, 2024-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43276.png

Downloads & links

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Map

FileDownload / File: emd_43276.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationInhibited state 2 full map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.93 Å/pix.
x 400 pix.
= 372. Å		0.93 Å/pix.
x 400 pix.
= 372. Å		0.93 Å/pix.
x 400 pix.
= 372. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.93 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.12
Minimum - Maximum-0.28381073 - 0.44131744
Average (Standard dev.)0.0019580105 (±0.01473003)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 372.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: LBD TMD local map

Fileemd_43276_additional_1.map
AnnotationLBD TMD local map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: TMD local map

Fileemd_43276_additional_2.map
AnnotationTMD local map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Inhibited state 2 full half B

Fileemd_43276_half_map_1.map
AnnotationInhibited state 2 full half B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Inhibited state 2 full half A

Fileemd_43276_half_map_2.map
AnnotationInhibited state 2 full half A
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : AMPA Receptor GluA2 Homotetramer

EntireName: AMPA Receptor GluA2 Homotetramer
Components
  • Complex: AMPA Receptor GluA2 Homotetramer
    • Protein or peptide: Isoform Flip of Glutamate receptor 2
  • Ligand: GLUTAMIC ACID
  • Ligand: 4-[(5S,8R)-8-methyl-6,7,8,9-tetrahydro-2H,5H-[1,3]dioxolo[4,5-h][2,3]benzodiazepin-5-yl]aniline

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Supramolecule #1: AMPA Receptor GluA2 Homotetramer

SupramoleculeName: AMPA Receptor GluA2 Homotetramer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Rattus norvegicus (Norway rat)

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Macromolecule #1: Isoform Flip of Glutamate receptor 2

MacromoleculeName: Isoform Flip of Glutamate receptor 2 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 89.232961 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: NSIQIGGLFP RGADQEYSAF RVGMVQFSTS EFRLTPHIDN LEVANSFAVT NAFCSQFSRG VYAIFGFYDK KSVNTITSFC GTLHVSFIT PSFPTDGTHP FVIQMRPDLK GALLSLIEYY QWDKFAYLYD SDRGLSTLQA VLDSAAEKKW QVTAINVGNI N NDKKDETY ...String:
NSIQIGGLFP RGADQEYSAF RVGMVQFSTS EFRLTPHIDN LEVANSFAVT NAFCSQFSRG VYAIFGFYDK KSVNTITSFC GTLHVSFIT PSFPTDGTHP FVIQMRPDLK GALLSLIEYY QWDKFAYLYD SDRGLSTLQA VLDSAAEKKW QVTAINVGNI N NDKKDETY RSLFQDLELK KERRVILDCE RDKVNDIVDQ VITIGKHVKG YHYIIANLGF TDGDLLKIQF GGAEVSGFQI VD YDDSLVS KFIERWSTLE EKEYPGAHTA TIKYTSALTY DAVQVMTEAF RNLRKQRIEI SRRGNAGDCL ANPAVPWGQG VEI ERALKQ VQVEGLSGNI KFDQNGKRIN YTINIMELKT NGPRKIGYWS EVDKMVLTED DTSGLEQKTV VVTTILESPY VMMK KNHEM LEGNERYEGY CVDLAAEIAK HCGFKYKLTI VGDGKYGARD ADTKIWNGMV GELVYGKADI AIAPLTITLV REEVI DFSK PFMSLGISIM IKKPQKSKPG VFSFLDPLAY EIWMCIVFAY IGVSVVLFLV SRFSPYSEST NEFGIFNSLW FSLGAF MQQ GCDISPRSLS GRIVGGVWWF FTLIIISSYT ANLAAFLTVE RMVSPIESAE DLSKQTEIAY GTLDSGSTKE FFRRSKI AV FDKMWTYMRS AEPSVFVRTT AEGVARVRKS KGKYAYLLES TMNEYIEQRK PCDTMKVGGN LDSKGYGIAT PKGSSLGT P VNLAVLKLSE QGVLDKLKNK WWYDKGECGA KDSGSKEKTS ALSLSNVAGV FYILVGGLGL AMLVALIEFC YKSRAE

UniProtKB: Glutamate receptor 2

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Macromolecule #2: GLUTAMIC ACID

MacromoleculeName: GLUTAMIC ACID / type: ligand / ID: 2 / Number of copies: 4 / Formula: GLU
Molecular weightTheoretical: 147.129 Da
Chemical component information

ChemComp-GLU:
GLUTAMIC ACID

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Macromolecule #3: 4-[(5S,8R)-8-methyl-6,7,8,9-tetrahydro-2H,5H-[1,3]dioxolo[4,5-h][...

MacromoleculeName: 4-[(5S,8R)-8-methyl-6,7,8,9-tetrahydro-2H,5H-[1,3]dioxolo[4,5-h][2,3]benzodiazepin-5-yl]aniline
type: ligand / ID: 3 / Number of copies: 4 / Formula: A1AB5
Molecular weightTheoretical: 293.32 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 2.6 µm / Calibrated defocus min: 1.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: ab initio
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.85 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 130474
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC

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