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- EMDB-7962: Open state GluA2 in complex with STZ and blocked by AgTx-636, aft... -

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Basic information

Entry
Database: EMDB / ID: EMD-7962
TitleOpen state GluA2 in complex with STZ and blocked by AgTx-636, after micelle signal subtraction
Map dataTMD-directed refinement, micelle signal subtracted
Sample
  • Complex: Open state GluA2 in complex with STZ and blocked by AgTx-636, after micelle signal subtraction
    • Protein or peptide: Glutamate receptor 2,Voltage-dependent calcium channel gamma-2 subunit
  • Ligand: GLUTAMIC ACID
  • Ligand: CYCLOTHIAZIDE
  • Ligand: N~1~-{5-[(3-{[3-(D-arginylamino)propyl]amino}propyl)amino]pentyl}-N~2~-[(2,4-dihydroxyphenyl)acetyl]-D-aspartamide
Function / homology
Function and homology information


LGI-ADAM interactions / Presynaptic depolarization and calcium channel opening / eye blink reflex / positive regulation of protein localization to basolateral plasma membrane / Trafficking of AMPA receptors / cerebellar mossy fiber / postsynaptic neurotransmitter receptor diffusion trapping / regulation of AMPA receptor activity / channel regulator activity / protein targeting to membrane ...LGI-ADAM interactions / Presynaptic depolarization and calcium channel opening / eye blink reflex / positive regulation of protein localization to basolateral plasma membrane / Trafficking of AMPA receptors / cerebellar mossy fiber / postsynaptic neurotransmitter receptor diffusion trapping / regulation of AMPA receptor activity / channel regulator activity / protein targeting to membrane / voltage-gated calcium channel complex / regulation of synaptic plasticity by chemical substance / spine synapse / dendritic spine neck / dendritic spine cytoplasm / response to phorbol 13-acetate 12-myristate / cellular response to amine stimulus / dendritic spine head / neurotransmitter receptor localization to postsynaptic specialization membrane / Activation of AMPA receptors / ligand-gated monoatomic cation channel activity / perisynaptic space / response to lithium ion / Trafficking of GluR2-containing AMPA receptors / AMPA glutamate receptor activity / AMPA glutamate receptor clustering / regulation of receptor recycling / kainate selective glutamate receptor activity / transmission of nerve impulse / AMPA glutamate receptor complex / extracellularly glutamate-gated ion channel activity / ionotropic glutamate receptor complex / cellular response to glycine / immunoglobulin binding / asymmetric synapse / Unblocking of NMDA receptors, glutamate binding and activation / glutamate receptor binding / positive regulation of synaptic transmission / conditioned place preference / regulation of synaptic transmission, glutamatergic / response to fungicide / voltage-gated calcium channel activity / extracellular ligand-gated monoatomic ion channel activity / cytoskeletal protein binding / glutamate-gated receptor activity / positive regulation of synaptic transmission, glutamatergic / cellular response to brain-derived neurotrophic factor stimulus / regulation of long-term synaptic depression / glutamate-gated calcium ion channel activity / somatodendritic compartment / presynaptic active zone membrane / ionotropic glutamate receptor binding / ionotropic glutamate receptor signaling pathway / excitatory synapse / dendrite cytoplasm / ligand-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / dendrite membrane / hippocampal mossy fiber to CA3 synapse / positive regulation of excitatory postsynaptic potential / SNARE binding / dendritic shaft / synaptic membrane / establishment of protein localization / synaptic transmission, glutamatergic / PDZ domain binding / protein tetramerization / long-term synaptic potentiation / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / receptor internalization / cerebral cortex development / response to calcium ion / postsynaptic density membrane / modulation of chemical synaptic transmission / Schaffer collateral - CA1 synapse / terminal bouton / endocytic vesicle membrane / synaptic vesicle membrane / synaptic vesicle / amyloid-beta binding / presynapse / signaling receptor activity / growth cone / chemical synaptic transmission / scaffold protein binding / presynaptic membrane / dendritic spine / perikaryon / neuron projection / postsynaptic membrane / postsynaptic density / external side of plasma membrane / axon / neuronal cell body / dendrite / protein kinase binding / synapse / protein-containing complex binding / glutamatergic synapse / perinuclear region of cytoplasm / cell surface
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 / 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 ...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 / 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 / Voltage-dependent calcium channel gamma-2 subunit
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsTwomey EC / Yelshanskaya MV / Vassilevski AA / Sobolevsky AI
CitationJournal: Neuron / Year: 2018
Title: Mechanisms of Channel Block in Calcium-Permeable AMPA Receptors.
Authors: Edward C Twomey / Maria V Yelshanskaya / Alexander A Vassilevski / Alexander I Sobolevsky /
Abstract: AMPA receptors mediate fast excitatory neurotransmission and are critical for CNS development and function. Calcium-permeable subsets of AMPA receptors are strongly implicated in acute and chronic ...AMPA receptors mediate fast excitatory neurotransmission and are critical for CNS development and function. Calcium-permeable subsets of AMPA receptors are strongly implicated in acute and chronic neurological disorders. However, despite the clinical importance, the therapeutic landscape for specifically targeting them, and not the calcium-impermeable AMPA receptors, remains largely undeveloped. To address this problem, we used cryo-electron microscopy and electrophysiology to investigate the mechanisms by which small-molecule blockers selectively inhibit ion channel conductance in calcium-permeable AMPA receptors. We determined the structures of calcium-permeable GluA2 AMPA receptor complexes with the auxiliary subunit stargazin bound to channel blockers, including the orb weaver spider toxin AgTx-636, the spider toxin analog NASPM, and the adamantane derivative IEM-1460. Our structures provide insights into the architecture of the blocker binding site and the mechanism of trapping, which are critical for development of small molecules that specifically target calcium-permeable AMPA receptors.
History
DepositionJun 4, 2018-
Header (metadata) releaseJul 4, 2018-
Map releaseAug 22, 2018-
UpdateSep 19, 2018-
Current statusSep 19, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6o9g
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7962.png

Downloads & links

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Map

FileDownload / File: emd_7962.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTMD-directed refinement, micelle signal subtracted
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 300 pix.
= 324. Å		1.08 Å/pix.
x 300 pix.
= 324. Å		1.08 Å/pix.
x 300 pix.
= 324. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.045 / Movie #1: 0.045
Minimum - Maximum-0.06412086 - 0.12932764
Average (Standard dev.)0.00062548835 (±0.005142609)
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-0.0640.1290.001

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Supplemental data

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Additional map: Full map, micelle signal subtracted

Fileemd_7962_additional.map
AnnotationFull map, micelle signal subtracted
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Open state GluA2 in complex with STZ and blocked by AgTx-636, aft...

EntireName: Open state GluA2 in complex with STZ and blocked by AgTx-636, after micelle signal subtraction
Components
  • Complex: Open state GluA2 in complex with STZ and blocked by AgTx-636, after micelle signal subtraction
    • Protein or peptide: Glutamate receptor 2,Voltage-dependent calcium channel gamma-2 subunit
  • Ligand: GLUTAMIC ACID
  • Ligand: CYCLOTHIAZIDE
  • Ligand: N~1~-{5-[(3-{[3-(D-arginylamino)propyl]amino}propyl)amino]pentyl}-N~2~-[(2,4-dihydroxyphenyl)acetyl]-D-aspartamide

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Supramolecule #1: Open state GluA2 in complex with STZ and blocked by AgTx-636, aft...

SupramoleculeName: Open state GluA2 in complex with STZ and blocked by AgTx-636, after micelle signal subtraction
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: The full-map (non-TMD-directed) is included as a supplemental file
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 subunit

MacromoleculeName: Glutamate receptor 2,Voltage-dependent calcium channel gamma-2 subunit
type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 115.178531 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 GAE

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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: 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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Macromolecule #4: N~1~-{5-[(3-{[3-(D-arginylamino)propyl]amino}propyl)amino]pentyl}...

MacromoleculeName: N~1~-{5-[(3-{[3-(D-arginylamino)propyl]amino}propyl)amino]pentyl}-N~2~-[(2,4-dihydroxyphenyl)acetyl]-D-aspartamide
type: ligand / ID: 4 / Number of copies: 1 / Formula: GYD
Molecular weightTheoretical: 636.787 Da
Chemical component information

ChemComp-GYD:
N~1~-{5-[(3-{[3-(D-arginylamino)propyl]amino}propyl)amino]pentyl}-N~2~-[(2,4-dihydroxyphenyl)acetyl]-D-aspartamide

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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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV
DetailsOpen state GluA2 in complex with STZ and blocked by AgTx-636, after micelle signal subtraction

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Electron microscopy

MicroscopeFEI TITAN KRIOS
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD
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


Details: Activated GluA2 complex bound to glutamate, cyclothiazide, and STZ in digitonin
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF
Details: Resolution for full map (supplemental file) is 4.8 angstrom.
Number images used: 82131
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-6o9g:
Open state GluA2 in complex with STZ and blocked by AgTx-636, after micelle signal subtraction

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