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- EMDB-29380: LBD conformation 1 (LBDconf1) of GluA2 flip Q isoform of AMPA rec... -

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Basic information

Entry
Database: EMDB / ID: EMD-29380
TitleLBD conformation 1 (LBDconf1) of GluA2 flip Q isoform of AMPA receptor in complex with gain-of-function TARP gamma2, with 140mM NMDG, 330uM CTZ, and 100mM L-glutamate (Open-Na110)
Map dataLBDconf1, Open-NMDG
Sample
  • Complex: Homotetrameric assembly of GluA2 flip-Q isoform in complex with TARP gamma2 (K52E, K53E). Dimer-of-dimers assembly.
    • Protein or peptide: Glutamate receptor 2GRIA2
  • Ligand: GLUTAMIC ACID
  • Ligand: CYCLOTHIAZIDE
  • Ligand: water
KeywordsInotropic glutamate receptors / AMPA receptors / ligand gated ion channel / auxiliary subunit / TARP / stargazin / TARP gamma2 / glutamate / calcium / neurotransmitter receptor / synaptic transmission / TRANSPORT PROTEIN
Function / homology
Function and homology information


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 ...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 / positive regulation of synaptic transmission / glutamate-gated receptor activity / presynaptic active zone membrane / response to fungicide / 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 / ionotropic glutamate receptor signaling pathway / dendrite cytoplasm / cytoskeletal protein binding / SNARE binding / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / dendritic shaft / 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 / presynaptic membrane / signaling receptor activity / amyloid-beta binding / growth cone / perikaryon / chemical synaptic transmission / scaffold protein binding / postsynaptic membrane / dendritic spine / postsynaptic density / neuron projection / axon / neuronal cell body / dendrite / synapse / glutamatergic 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
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.11 Å
AuthorsNakagawa T
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)MH123474 United States
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: The open gate of the AMPA receptor forms a Ca binding site critical in regulating ion transport.
Authors: Terunaga Nakagawa / Xin-Tong Wang / Federico J Miguez-Cabello / Derek Bowie /
Abstract: Alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid receptors (AMPARs) are cation-selective ion channels that mediate most fast excitatory neurotransmission in the brain. Although their gating ...Alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid receptors (AMPARs) are cation-selective ion channels that mediate most fast excitatory neurotransmission in the brain. Although their gating mechanism has been studied extensively, understanding how cations traverse the pore has remained elusive. Here we investigated putative ion and water densities in the open pore of Ca-permeable AMPARs (rat GRIA2 flip-Q isoform) at 2.3-2.6 Å resolution. We show that the ion permeation pathway attains an extracellular Ca binding site (site-G) when the channel gate moves into the open configuration. Site-G is highly selective for Ca over Na, favoring the movement of Ca into the selectivity filter of the pore. Seizure-related N619K mutation, adjacent to site-G, promotes channel opening but attenuates Ca binding and thus diminishes Ca permeability. Our work identifies the importance of site-G, which coordinates with the Q/R site of the selectivity filter to ensure the preferential transport of Ca through the channel pore.
History
DepositionJan 5, 2023-
Header (metadata) releaseFeb 28, 2024-
Map releaseFeb 28, 2024-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29380.png

Downloads & links

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Map

FileDownload / File: emd_29380.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLBDconf1, Open-NMDG
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 360 pix.
= 295.02 Å		0.82 Å/pix.
x 360 pix.
= 295.02 Å		0.82 Å/pix.
x 360 pix.
= 295.02 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8195 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.013
Minimum - Maximum-0.04118036 - 0.07517466
Average (Standard dev.)0.00002487955 (±0.0022141398)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 295.02002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_29380_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half1 map, Open-NMDG

Fileemd_29380_half_map_1.map
AnnotationHalf1 map, Open-NMDG
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half2 map, Open-NMDG

Fileemd_29380_half_map_2.map
AnnotationHalf2 map, Open-NMDG
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Homotetrameric assembly of GluA2 flip-Q isoform in complex with T...

EntireName: Homotetrameric assembly of GluA2 flip-Q isoform in complex with TARP gamma2 (K52E, K53E). Dimer-of-dimers assembly.
Components
  • Complex: Homotetrameric assembly of GluA2 flip-Q isoform in complex with TARP gamma2 (K52E, K53E). Dimer-of-dimers assembly.
    • Protein or peptide: Glutamate receptor 2GRIA2
  • Ligand: GLUTAMIC ACID
  • Ligand: CYCLOTHIAZIDE
  • Ligand: water

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Supramolecule #1: Homotetrameric assembly of GluA2 flip-Q isoform in complex with T...

SupramoleculeName: Homotetrameric assembly of GluA2 flip-Q isoform in complex with TARP gamma2 (K52E, K53E). Dimer-of-dimers assembly.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 500 KDa

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Macromolecule #1: Glutamate receptor 2

MacromoleculeName: Glutamate receptor 2 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 99.559461 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MQKIMHISVL LSPVLWGLIF GVSSNSIQIG GLFPRGADQE YSAFRVGMVQ FSTSEFRLTP HIDNLEVANS FAVTNAFCSQ FSRGVYAIF GFYDKKSVNT ITSFCGTLHV SFITPSFPTD GTHPFVIQMR PDLKGALLSL IEYYQWDKFA YLYDSDRGLS T LQAVLDSA ...String:
MQKIMHISVL LSPVLWGLIF GVSSNSIQIG GLFPRGADQE YSAFRVGMVQ FSTSEFRLTP HIDNLEVANS FAVTNAFCSQ FSRGVYAIF GFYDKKSVNT ITSFCGTLHV SFITPSFPTD GTHPFVIQMR PDLKGALLSL IEYYQWDKFA YLYDSDRGLS T LQAVLDSA AEKKWQVTAI NVGNINNDKK DETYRSLFQD LELKKERRVI LDCERDKVND IVDQVITIGK HVKGYHYIIA NL GFTDGDL LKIQFGGANV SGFQIVDYDD SLVSKFIERW STLEEKEYPG AHTATIKYTS ALTYDAVQVM TEAFRNLRKQ RIE ISRRGN AGDCLANPAV PWGQGVEIER ALKQVQVEGL SGNIKFDQNG KRINYTINIM ELKTNGPRKI GYWSEVDKMV VTLT ELPSG NDTSGLENKT VVVTTILESP YVMMKKNHEM LEGNERYEGY CVDLAAEIAK HCGFKYKLTI VGDGKYGARD ADTKI WNGM VGELVYGKAD IAIAPLTITL VREEVIDFSK PFMSLGISIM IKKPQKSKPG VFSFLDPLAY EIWMCIVFAY IGVSVV LFL VSRFSPYEWH TEEFEDGRET QSSESTNEFG IFNSLWFSLG AFMQQGCDIS PRSLSGRIVG GVWWFFTLII ISSYTAN LA AFLTVERMVS PIESAEDLSK QTEIAYGTLD SGSTKEFFRR SKIAVFDKMW TYMRSAEPSV FVRTTAEGVA RVRKSKGK Y AYLLESTMNE YIEQRKPCDT MKVGGNLDSK GYGIATPKGS SLGTPVNLAV LKLSEQGVLD KLKNKWWYDK GECGAKDSG SKEKTSALSL SNVAGVFYIL VGGLGLAMLV ALIEFCYKSR AEAKRMKVAK NPQNINPSSS QNSQNFATDY KDDDDKEGYN VYGIESVKI

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 / Glutamic acid

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Macromolecule #3: CYCLOTHIAZIDE

MacromoleculeName: CYCLOTHIAZIDE / type: ligand / ID: 3 / Number of copies: 4 / Formula: CYZ
Molecular weightTheoretical: 389.878 Da
Chemical component information

ChemComp-CYZ:
CYCLOTHIAZIDE / Cyclothiazide

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 10 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

Concentration10 mg/mL
BufferpH: 8
Component:
ConcentrationName
20.0 mMTris-HClTris
140.0 mMN-methyl-D-glucamine
0.03 %GDN

Details: L-glutamic acid (100mM) and cyclothiazide (CTZ, 0.33mM) were added before freezing. The 1 M L-glutamic acid stock solution was adjusted to pH 7.4 using NaOH.
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 30384 / Average electron dose: 52.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

2680

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationNumber classes: 3 / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 189181
FSC plot (resolution estimation)

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