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- EMDB-8823: Activated GluA2 complex bound to glutamate, cyclothiazide, and ST... -

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Basic information

Entry
Database: EMDB / ID: EMD-8823
TitleActivated GluA2 complex bound to glutamate, cyclothiazide, and STZ in digitonin
Map dataActivated GluA2 complex bound to glutamate, cyclothiazide, and STZ in digitonin
Sample
  • Complex: Activated GluA2 complex bound to glutamate, cyclothiazide, and STZ in digitonin
    • Protein or peptide: Glutamate receptor 2,Voltage-dependent calcium channel gamma-2 subunit chimera
  • Ligand: GLUTAMIC ACID
  • Ligand: CYCLOTHIAZIDE
Function / homology
Function and homology information


Presynaptic depolarization and calcium channel opening / LGI-ADAM interactions / regulation of postsynaptic neurotransmitter receptor activity / Trafficking of AMPA receptors / eye blink reflex / positive regulation of protein localization to basolateral plasma membrane / cerebellar mossy fiber / neurotransmitter receptor transport, postsynaptic endosome to lysosome / regulation of AMPA receptor activity / neurotransmitter receptor internalization ...Presynaptic depolarization and calcium channel opening / LGI-ADAM interactions / regulation of postsynaptic neurotransmitter receptor activity / Trafficking of AMPA receptors / eye blink reflex / positive regulation of protein localization to basolateral plasma membrane / cerebellar mossy fiber / neurotransmitter receptor transport, postsynaptic endosome to lysosome / regulation of AMPA receptor activity / neurotransmitter receptor internalization / channel regulator activity / membrane hyperpolarization / postsynaptic neurotransmitter receptor diffusion trapping / nervous system process / protein targeting to membrane / neurotransmitter receptor localization to postsynaptic specialization membrane / neuromuscular junction development / spine synapse / dendritic spine neck / voltage-gated calcium channel complex / dendritic spine head / Activation of AMPA receptors / response to lithium ion / cellular response to glycine / perisynaptic space / transmission of nerve impulse / AMPA glutamate receptor activity / regulation of postsynaptic membrane neurotransmitter receptor levels / Trafficking of GluR2-containing AMPA receptors / membrane depolarization / 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 / voltage-gated calcium channel activity / glutamate receptor binding / 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 / hippocampal mossy fiber to CA3 synapse / cytoskeletal protein binding / regulation of membrane potential / ionotropic glutamate receptor signaling pathway / dendrite cytoplasm / positive regulation of synaptic transmission, glutamatergic / 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 / response to calcium ion / 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 / neuronal cell body / dendrite / 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
Voltage-dependent calcium channel, gamma-2 subunit / PMP-22/EMP/MP20/Claudin family / Voltage-dependent calcium channel, gamma subunit / PMP-22/EMP/MP20/Claudin superfamily / 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 ...Voltage-dependent calcium channel, gamma-2 subunit / PMP-22/EMP/MP20/Claudin family / Voltage-dependent calcium channel, gamma subunit / PMP-22/EMP/MP20/Claudin superfamily / 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
Voltage-dependent calcium channel gamma-2 subunit / Glutamate receptor 2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsTwomey EC / Yelshanskaya MV / Grassucci RA / Frank J / Sobolevsky AI
Funding support United States, 5 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS093838 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA206573 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS083660 United States
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM029169 United States
CitationJournal: Nature / Year: 2017
Title: Channel opening and gating mechanism in AMPA-subtype glutamate receptors.
Authors: Edward C Twomey / Maria V Yelshanskaya / Robert A Grassucci / Joachim Frank / Alexander I Sobolevsky /
Abstract: AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-subtype ionotropic glutamate receptors mediate fast excitatory neurotransmission throughout the central nervous system. Gated by the ...AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-subtype ionotropic glutamate receptors mediate fast excitatory neurotransmission throughout the central nervous system. Gated by the neurotransmitter glutamate, AMPA receptors are critical for synaptic strength, and dysregulation of AMPA receptor-mediated signalling is linked to numerous neurological diseases. Here we use cryo-electron microscopy to solve the structures of AMPA receptor-auxiliary subunit complexes in the apo, antagonist- and agonist-bound states and determine the iris-like mechanism of ion channel opening. The ion channel selectivity filter is formed by the extended portions of the re-entrant M2 loops, while the helical portions of M2 contribute to extensive hydrophobic interfaces between AMPA receptor subunits in the ion channel. We show how the permeation pathway changes upon channel opening and identify conformational changes throughout the entire AMPA receptor that accompany activation and desensitization. Our findings provide a framework for understanding gating across the family of ionotropic glutamate receptors and the role of AMPA receptors in excitatory neurotransmission.
History
DepositionJul 10, 2017-
Header (metadata) releaseAug 2, 2017-
Map releaseAug 2, 2017-
UpdateNov 20, 2019-
Current statusNov 20, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5weo
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8823.png

Downloads & links

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Map

FileDownload / File: emd_8823.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationActivated GluA2 complex bound to glutamate, cyclothiazide, and STZ in digitonin
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.09160006 - 0.16419825
Average (Standard dev.)0.0006511989 (±0.0055219787)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 324.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z324.000324.000324.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0920.1640.001

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Supplemental data

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Additional map: Focused TMD refinement of activated complex of GluA2...

Fileemd_8823_additional.map
AnnotationFocused TMD refinement of activated complex of GluA2 bound to glutamate, cyclothiazide and STZ
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Activated GluA2 complex bound to glutamate, cyclothiazide, and ST...

EntireName: Activated GluA2 complex bound to glutamate, cyclothiazide, and STZ in digitonin
Components
  • Complex: Activated GluA2 complex bound to glutamate, cyclothiazide, and STZ in digitonin
    • Protein or peptide: Glutamate receptor 2,Voltage-dependent calcium channel gamma-2 subunit chimera
  • Ligand: GLUTAMIC ACID
  • Ligand: CYCLOTHIAZIDE

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Supramolecule #1: Activated GluA2 complex bound to glutamate, cyclothiazide, and ST...

SupramoleculeName: Activated GluA2 complex bound to glutamate, cyclothiazide, and STZ in digitonin
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Rattus norvegicus (Norway rat)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293 gnti-

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Macromolecule #1: Glutamate receptor 2,Voltage-dependent calcium channel gamma-2 su...

MacromoleculeName: Glutamate receptor 2,Voltage-dependent calcium channel gamma-2 subunit chimera
type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 115.501969 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 GLVPRG

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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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration4 mg/mL
BufferpH: 8
GridModel: C-flat-1.2/1.3 / Material: GOLD / Mesh: 200
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV
DetailsActivated GluA2 complex bound to glutamate, cyclothiazide, and STZ in digitonin

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 8.0 sec. / Average electron dose: 55.0 e/Å2
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:

5kbu


Details: GluA2-2xSTZ in DDM
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF
Details: TMD map uploaded under additional files is at 4.0 angstrom resolution.
Number images used: 69207

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-5weo:
Activated GluA2 complex bound to glutamate, cyclothiazide, and STZ in digitonin

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