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- EMDB-15985: Small molecule positive allosteric modulation of homomeric kainat... -

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Basic information

Entry
Database: EMDB / ID: EMD-15985
TitleSmall molecule positive allosteric modulation of homomeric kainate receptors GluK1-3: Development of screening assays and insight into GluK3 structure
Map dataMsp
Sample
  • Complex: Complex of GRIK3_HUMAN with glutamate
    • Protein or peptide: GRIK3_HUMAN
Keywordsionotropic glutamate receptor / MEMBRANE PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 6.6 Å
AuthorsGajhede M / Boesen T
Funding support Denmark, 2 items
OrganizationGrant numberCountry
Lundbeckfonden Denmark
Other government Denmark
CitationJournal: FEBS J / Year: 2024
Title: Small-molecule positive allosteric modulation of homomeric kainate receptors GluK1-3: development of screening assays and insight into GluK3 structure.
Authors: Yasmin Bay / Raminta Venskutonytė / Stine M Frantsen / Thor S Thorsen / Maria Musgaard / Karla Frydenvang / Pierre Francotte / Bernard Pirotte / Philip C Biggin / Anders S Kristensen / ...Authors: Yasmin Bay / Raminta Venskutonytė / Stine M Frantsen / Thor S Thorsen / Maria Musgaard / Karla Frydenvang / Pierre Francotte / Bernard Pirotte / Philip C Biggin / Anders S Kristensen / Thomas Boesen / Darryl S Pickering / Michael Gajhede / Jette S Kastrup /
Abstract: The kainate receptors GluK1-3 (glutamate receptor ionotropic, kainate receptors 1-3) belong to the family of ionotropic glutamate receptors and are essential for fast excitatory neurotransmission in ...The kainate receptors GluK1-3 (glutamate receptor ionotropic, kainate receptors 1-3) belong to the family of ionotropic glutamate receptors and are essential for fast excitatory neurotransmission in the brain, and are associated with neurological and psychiatric diseases. How these receptors can be modulated by small-molecule agents is not well understood, especially for GluK3. We show that the positive allosteric modulator BPAM344 can be used to establish robust calcium-sensitive fluorescence-based assays to test agonists, antagonists, and positive allosteric modulators of GluK1-3. The half-maximal effective concentration (EC) of BPAM344 for potentiating the response of 100 μm kainate was determined to be 26.3 μm for GluK1, 75.4 μm for GluK2, and 639 μm for GluK3. Domoate was found to be a potent agonist for GluK1 and GluK2, with an EC of 0.77 and 1.33 μm, respectively, upon co-application of 150 μm BPAM344. At GluK3, domoate acts as a very weak agonist or antagonist with a half-maximal inhibitory concentration (IC) of 14.5 μm, in presence of 500 μm BPAM344 and 100 μm kainate for competition binding. Using H523A-mutated GluK3, we determined the first dimeric structure of the ligand-binding domain by X-ray crystallography, allowing location of BPAM344, as well as zinc-, sodium-, and chloride-ion binding sites at the dimer interface. Molecular dynamics simulations support the stability of the ion sites as well as the involvement of Asp761, Asp790, and Glu797 in the binding of zinc ions. Using electron microscopy, we show that, in presence of glutamate and BPAM344, full-length GluK3 adopts a dimer-of-dimers arrangement.
History
DepositionOct 20, 2022-
Header (metadata) releaseNov 1, 2023-
Map releaseNov 1, 2023-
UpdateApr 10, 2024-
Current statusApr 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15985.png

Downloads & links

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Map

FileDownload / File: emd_15985.map.gz / Format: CCP4 / Size: 3.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMsp
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.14 Å/pix.
x 100 pix.
= 314. Å		3.14 Å/pix.
x 100 pix.
= 314. Å		3.14 Å/pix.
x 100 pix.
= 314. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.14 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.11
Minimum - Maximum-0.63509345 - 3.9881403
Average (Standard dev.)-0.00011797196 (±0.16824052)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 314.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map

Fileemd_15985_half_map_1.map
AnnotationHalf map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map

Fileemd_15985_half_map_2.map
AnnotationHalf map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of GRIK3_HUMAN with glutamate

EntireName: Complex of GRIK3_HUMAN with glutamate
Components
  • Complex: Complex of GRIK3_HUMAN with glutamate
    • Protein or peptide: GRIK3_HUMAN

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Supramolecule #1: Complex of GRIK3_HUMAN with glutamate

SupramoleculeName: Complex of GRIK3_HUMAN with glutamate / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: GRIK3_HUMAN

MacromoleculeName: GRIK3_HUMAN / type: protein_or_peptide / ID: 1 / Enantiomer: DEXTRO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MTAPWRRLRS LVWEYWAGLL VCAFWIPDSR GMPHVIRIGG IFEYADGPNA QVMNAEEHAF RFSANIINRN RTLLPNTTLT YDIQRIHFHD SFEATKKACD QLALGVVAIF GPSQGSCTNA VQSICNALEV PHIQLRWKHH PLDNKDTFYV NLYPDYASLS HAILDLVQYL ...String:
MTAPWRRLRS LVWEYWAGLL VCAFWIPDSR GMPHVIRIGG IFEYADGPNA QVMNAEEHAF RFSANIINRN RTLLPNTTLT YDIQRIHFHD SFEATKKACD QLALGVVAIF GPSQGSCTNA VQSICNALEV PHIQLRWKHH PLDNKDTFYV NLYPDYASLS HAILDLVQYL KWRSATVVYD DSTGLIRLQE LIMAPSRYNI RLKIRQLPID SDDSRPLLKE MKRGREFRII FDCSHTMAAQ ILKQAMAMGM MTEYYHFIFT TLDLYALDLE PYRYSGVNLT GFRILNVDNP HVSAIVEKWS MERLQAAPRS ESGLLDGVMM TDAALLYDAV HIVSVCYQRA PQMTVNSLQC HRHKAWRFGG RFMNFIKEAQ WEGLTGRIVF NKTSGLRTDF DLDIISLKED GLEKVGVWSP ADGLNITEVA KGRGPNVTDS LTNRSLIVTT VLEEPFVMFR KSDRTLYGND RFEGYCIDLL KELAHILGFS YEIRLVEDGK YGAQDDKGQW NGMVKELIDH KADLAVAPLT ITHVREKAID FSKPFMTLGV SILYRKPNGT NPSVFSFLNP LSPDIWMYVL LAYLGVSCVL FVIARFSPYE WYDAHPCNPG SEVVENNFTL LNSFWFGMGS LMQQGSELMP KALSTRIIGG IWWFFTLIII SSYTANLAAF LTVERMESPI DSADDLAKQT KIEYGAVKDG ATMTFFKKSK ISTFEKMWAF MSSKPSALVK NNEEGIQRAL TADYALLMES TTIEYVTQRN CNLTQIGGLI DSKGYGIGTP MGSPYRDKIT IAILQLQEED KLHIMKEKWW RGSGCPEEEN KEASALGIQK IGGIFIVLAA GLVLSVLVAV GEFVYKLRKT AEREQRSFCS TVADEIRFSL TCQRRVKHKP QPPMMVKTDA VINMHTFNDR RLPGKDSMAC STSLAPVFP

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
StainingType: NEGATIVE / Material: Uranyl formate
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Image recordingFilm or detector model: OTHER / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 82515
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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