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- EMDB-26016: Complex GGGN of AMPA-subtype iGluR GluA2 in complex with auxiliar... -

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Basic information

Entry
Database: EMDB / ID: EMD-26016
TitleComplex GGGN of AMPA-subtype iGluR GluA2 in complex with auxiliary subunit gamma2 (Stargazin) at low glutamate concentration (20 uM) in the presence of cyclothiazide (100 uM)
Map dataComplex GGGN of AMPA-subtype iGluR GluA2 in complex with auxiliary subunit gamma2 (Stargazin) at low glutamate concentration (20 uM) in the presence of cyclothiazide (100 uM)
Sample
  • Complex: Ionotropic glutamate receptor GluA2 in complex with auxiliary subunit gamma2 or Stargazin
    • Protein or peptide: Isoform Flip of Glutamate receptor 2,Voltage-dependent calcium channel gamma-3 subunit chimera
  • Ligand: GLUTAMIC ACID
  • Ligand: CYCLOTHIAZIDE
KeywordsAMPA / iGluR / Stargazin / cryo-EM / complex / agonist / positive allosteric modulator / subconductance level / opening / gating / glutamate / MEMBRANE PROTEIN
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 / extracellularly glutamate-gated ion channel activity / cellular response to glycine / asymmetric synapse / regulation of receptor recycling / Unblocking of NMDA receptors, glutamate binding and activation / positive regulation of synaptic transmission / glutamate receptor binding / extracellular ligand-gated monoatomic ion channel activity / glutamate-gated receptor activity / response to fungicide / glutamate-gated calcium ion channel activity / presynaptic active zone membrane / regulation of synaptic transmission, glutamatergic / somatodendritic compartment / dendrite membrane / cellular response to brain-derived neurotrophic factor stimulus / cytoskeletal protein binding / ligand-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / ionotropic glutamate receptor binding / dendrite cytoplasm / ionotropic glutamate receptor signaling pathway / SNARE binding / dendritic shaft / synaptic transmission, glutamatergic / synaptic membrane / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / PDZ domain binding / protein tetramerization / postsynaptic density membrane / establishment of protein localization / modulation of chemical synaptic transmission / Schaffer collateral - CA1 synapse / terminal bouton / receptor internalization / cerebral cortex development / synaptic vesicle membrane / synaptic vesicle / presynapse / signaling receptor activity / presynaptic membrane / amyloid-beta binding / growth cone / scaffold protein binding / chemical synaptic transmission / perikaryon / postsynaptic membrane / dendritic spine / postsynaptic density / neuron projection / axon / neuronal cell body / glutamatergic synapse / dendrite / synapse / protein-containing complex binding / protein kinase binding / 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: 4.02 Å
AuthorsYelshanskaya MV / Sobolevsky AI
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS083660 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS107253 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA206573 United States
National Science Foundation (NSF, United States)1818086 United States
CitationJournal: Nature / Year: 2022
Title: Opening of glutamate receptor channel to subconductance levels.
Authors: Maria V Yelshanskaya / Dhilon S Patel / Christopher M Kottke / Maria G Kurnikova / Alexander I Sobolevsky /
Abstract: Ionotropic glutamate receptors (iGluRs) are tetrameric ligand-gated ion channels that open their pores in response to binding of the agonist glutamate. An ionic current through a single iGluR channel ...Ionotropic glutamate receptors (iGluRs) are tetrameric ligand-gated ion channels that open their pores in response to binding of the agonist glutamate. An ionic current through a single iGluR channel shows up to four discrete conductance levels (O1-O4). Higher conductance levels have been associated with an increased number of agonist molecules bound to four individual ligand-binding domains (LBDs). Here we determine structures of a synaptic complex of AMPA-subtype iGluR and the auxiliary subunit γ2 in non-desensitizing conditions with various occupancy of the LBDs by glutamate. We show that glutamate binds to LBDs of subunits B and D only after it is already bound to at least the same number of LBDs that belong to subunits A and C. Our structures combined with single-channel recordings, molecular dynamics simulations and machine-learning analysis suggest that channel opening requires agonist binding to at least two LBDs. Conversely, agonist binding to all four LBDs does not guarantee maximal channel conductance and favours subconductance states O1 and O2, with O3 and O4 being rare and not captured structurally. The lack of subunit independence and low efficiency coupling of glutamate binding to channel opening underlie the gating of synaptic complexes to submaximal conductance levels, which provide a potential for upregulation of synaptic activity.
History
DepositionJan 21, 2022-
Header (metadata) releaseApr 20, 2022-
Map releaseApr 20, 2022-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26016.png

Downloads & links

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Map

FileDownload / File: emd_26016.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComplex GGGN of AMPA-subtype iGluR GluA2 in complex with auxiliary subunit gamma2 (Stargazin) at low glutamate concentration (20 uM) in the presence of cyclothiazide (100 uM)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 320 pix.
= 265.6 Å		0.83 Å/pix.
x 320 pix.
= 265.6 Å		0.83 Å/pix.
x 320 pix.
= 265.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015
Minimum - Maximum-0.04324271 - 0.075442374
Average (Standard dev.)0.00021545125 (±0.0022312535)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 265.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Ionotropic glutamate receptor GluA2 in complex with auxiliary sub...

EntireName: Ionotropic glutamate receptor GluA2 in complex with auxiliary subunit gamma2 or Stargazin
Components
  • Complex: Ionotropic glutamate receptor GluA2 in complex with auxiliary subunit gamma2 or Stargazin
    • Protein or peptide: Isoform Flip of Glutamate receptor 2,Voltage-dependent calcium channel gamma-3 subunit chimera
  • Ligand: GLUTAMIC ACID
  • Ligand: CYCLOTHIAZIDE

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Supramolecule #1: Ionotropic glutamate receptor GluA2 in complex with auxiliary sub...

SupramoleculeName: Ionotropic glutamate receptor GluA2 in complex with auxiliary subunit gamma2 or Stargazin
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,Voltage-dependent calcium ch...

MacromoleculeName: Isoform Flip of Glutamate receptor 2,Voltage-dependent calcium channel gamma-3 subunit chimera
type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 115.444922 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 SRFSPYEWHT EEFEDGRETQ SSESTN EFG IFNSLWFSLG AFMQQGCDIS PRSLSGRIVG GVWWFFTLII ISSYTANLAA FLTVERMVSP IESAEDLSKQ TEIAYGT LD SGSTKEFFRR SKIAVFDKMW TYMRSAEPSV FVRTTAEGVA RVRKSKGKYA YLLESTMNEY IEQRKPCDTM KVGGNLDS K GYGIATPKGS SLGTPVNLAV LKLSEQGVLD KLKNKWWYDK GECGAKDSGS KEKTSALSLS NVAGVFYILV GGLGLAMLV ALIEFCYKSR AEAKRMKGTG LFDRGVQMLL TTVGAFAAFS LMTIAVGTDY WLYSRGVCKT KSVSEDETSK KNEEVMTHSG LWRTCCLEG NFKGLCKQID HFPEDADYEA DTAEYFLRAV RASSIFPILS VILLFMGGLC IAASEFYKTR HNIILSAGIF F VSAGLSNI IGIIVYISAN AGDPSKSDSK KNSYSYGWSF YFGALSFIIA EMVGVLAVHM FIDRHKQLTG GLVPR

UniProtKB: Glutamate receptor 2

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Macromolecule #2: GLUTAMIC ACID

MacromoleculeName: GLUTAMIC ACID / type: ligand / ID: 2 / Number of copies: 3 / Formula: GLU
Molecular weightTheoretical: 147.129 Da
Chemical component information

ChemComp-GLU:
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

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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

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 58.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5weo

Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.02 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 51538
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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