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- EMDB-13970: Active state of GluA1/A2 AMPA receptor in complex with TARP gamma... -

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Basic information

Entry
Database: EMDB / ID: EMD-13970
TitleActive state of GluA1/A2 AMPA receptor in complex with TARP gamma-8 (TMD)
Map dataPostprocessed map of the TMD region of the complex GluA1/A2/TARP-gamma8
Sample
  • Complex: Complex between GluA1/2 AMPA receptor and auxiliary subunit TARP gamma8
    • Protein or peptide: AMPA Glutamate Receptor 1, isoform FLIP
    • Protein or peptide: AMPA Glutamate Receptor 2, isoform FLIP, Q/R edited
    • Protein or peptide: TARP gamma 8
Keywordsglutamate / AMPA receptor / TARPs / MEMBRANE PROTEIN
Function / homology
Function and homology information


Phase 2 - plateau phase / Phase 0 - rapid depolarisation / Cargo concentration in the ER / cellular response to amine stimulus / axonal spine / COPII-mediated vesicle transport / positive regulation of membrane potential / chemical synaptic transmission, postsynaptic / cellular response to ammonium ion / neurotransmitter receptor transport, postsynaptic endosome to lysosome ...Phase 2 - plateau phase / Phase 0 - rapid depolarisation / Cargo concentration in the ER / cellular response to amine stimulus / axonal spine / COPII-mediated vesicle transport / positive regulation of membrane potential / chemical synaptic transmission, postsynaptic / cellular response to ammonium ion / neurotransmitter receptor transport, postsynaptic endosome to lysosome / L-type voltage-gated calcium channel complex / neurotransmitter receptor activity involved in regulation of postsynaptic cytosolic calcium ion concentration / LGI-ADAM interactions / neuron spine / myosin V binding / Trafficking of AMPA receptors / regulation of AMPA receptor activity / neurotransmitter receptor internalization / channel regulator activity / protein phosphatase 2B binding / dendritic spine membrane / postsynaptic neurotransmitter receptor diffusion trapping / response to arsenic-containing substance / cellular response to dsRNA / Synaptic adhesion-like molecules / long-term synaptic depression / beta-2 adrenergic receptor binding / protein kinase A binding / cellular response to peptide hormone stimulus / neuronal cell body membrane / spine synapse / spinal cord development / dendritic spine neck / dendritic spine head / Activation of AMPA receptors / response to lithium ion / perisynaptic space / cellular response to glycine / transmission of nerve impulse / AMPA glutamate receptor activity / regulation of postsynaptic membrane neurotransmitter receptor levels / Trafficking of GluR2-containing AMPA receptors / immunoglobulin binding / calcium channel regulator activity / AMPA glutamate receptor complex / neuronal action potential / kainate selective glutamate receptor activity / excitatory synapse / ionotropic glutamate receptor complex / extracellularly glutamate-gated ion channel activity / adenylate cyclase binding / cellular response to organic cyclic compound / asymmetric synapse / G-protein alpha-subunit binding / regulation of receptor recycling / Unblocking of NMDA receptors, glutamate binding and activation / long-term memory / voltage-gated calcium channel activity / glutamate receptor binding / regulation of postsynaptic membrane potential / positive regulation of synaptic transmission / response to electrical stimulus / 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 / synapse assembly / 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 / monoatomic ion transmembrane transport / positive regulation of synaptic transmission, glutamatergic / SNARE binding / dendritic shaft / response to cocaine / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / synaptic membrane / synaptic transmission, glutamatergic / PDZ domain binding / cellular response to amino acid stimulus / postsynaptic density membrane / protein tetramerization / regulation of synaptic plasticity / modulation of chemical synaptic transmission / neuromuscular junction / Schaffer collateral - CA1 synapse / establishment of protein localization / terminal bouton / receptor internalization / synaptic vesicle membrane / response to organic cyclic compound / cerebral cortex development / response to peptide hormone / response to toxic substance / cellular response to growth factor stimulus
Similarity search - Function
Voltage-dependent calcium channel, gamma-8 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-8 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
Glutamate receptor 1 / Glutamate receptor 2 / Voltage-dependent calcium channel gamma-8 subunit
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.39 Å
AuthorsHerguedas B / Kohegyi B / Dohrke JN / Watson JF / Zhang D / Ho H / Shaikh SH / Lape R / Krieger JM / Greger IH
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_U105174197 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/N002113/1 United Kingdom
Spanish Ministry of Science, Innovation, and UniversitiesPID2019-106284GA-I00 United Kingdom
CitationJournal: Nat Commun / Year: 2022
Title: Mechanisms underlying TARP modulation of the GluA1/2-γ8 AMPA receptor.
Authors: Beatriz Herguedas / Bianka K Kohegyi / Jan-Niklas Dohrke / Jake F Watson / Danyang Zhang / Hinze Ho / Saher A Shaikh / Remigijus Lape / James M Krieger / Ingo H Greger /
Abstract: AMPA-type glutamate receptors (AMPARs) mediate rapid signal transmission at excitatory synapses in the brain. Glutamate binding to the receptor's ligand-binding domains (LBDs) leads to ion channel ...AMPA-type glutamate receptors (AMPARs) mediate rapid signal transmission at excitatory synapses in the brain. Glutamate binding to the receptor's ligand-binding domains (LBDs) leads to ion channel activation and desensitization. Gating kinetics shape synaptic transmission and are strongly modulated by transmembrane AMPAR regulatory proteins (TARPs) through currently incompletely resolved mechanisms. Here, electron cryo-microscopy structures of the GluA1/2 TARP-γ8 complex, in both open and desensitized states (at 3.5 Å), reveal state-selective engagement of the LBDs by the large TARP-γ8 loop ('β1'), elucidating how this TARP stabilizes specific gating states. We further show how TARPs alter channel rectification, by interacting with the pore helix of the selectivity filter. Lastly, we reveal that the Q/R-editing site couples the channel constriction at the filter entrance to the gate, and forms the major cation binding site in the conduction path. Our results provide a mechanistic framework of how TARPs modulate AMPAR gating and conductance.
History
DepositionDec 12, 2021-
Header (metadata) releaseFeb 23, 2022-
Map releaseFeb 23, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0275
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0275
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13970.png

Downloads & links

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Map

FileDownload / File: emd_13970.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostprocessed map of the TMD region of the complex GluA1/A2/TARP-gamma8
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 320 pix.
= 339.2 Å		1.06 Å/pix.
x 320 pix.
= 339.2 Å		1.06 Å/pix.
x 320 pix.
= 339.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0275 / Movie #1: 0.0275
Minimum - Maximum-0.0679944 - 0.12139431
Average (Standard dev.)0.00010019256 (±0.0019080449)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 339.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z339.200339.200339.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0680.1210.000

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Supplemental data

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Half map: Half map obtained in 3D refinement with Relion

Fileemd_13970_half_map_1.map
AnnotationHalf map obtained in 3D refinement with Relion
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map obtained in 3D refinement with Relion

Fileemd_13970_half_map_2.map
AnnotationHalf map obtained in 3D refinement with Relion
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 between GluA1/2 AMPA receptor and auxiliary subunit TARP ...

EntireName: Complex between GluA1/2 AMPA receptor and auxiliary subunit TARP gamma8
Components
  • Complex: Complex between GluA1/2 AMPA receptor and auxiliary subunit TARP gamma8
    • Protein or peptide: AMPA Glutamate Receptor 1, isoform FLIP
    • Protein or peptide: AMPA Glutamate Receptor 2, isoform FLIP, Q/R edited
    • Protein or peptide: TARP gamma 8

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Supramolecule #1: Complex between GluA1/2 AMPA receptor and auxiliary subunit TARP ...

SupramoleculeName: Complex between GluA1/2 AMPA receptor and auxiliary subunit TARP gamma8
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: GluA2 and TARP8 are expressed as a tandem construct
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 490 KDa

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Macromolecule #1: AMPA Glutamate Receptor 1, isoform FLIP

MacromoleculeName: AMPA Glutamate Receptor 1, isoform FLIP / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MPYIFAFFCT GFLGAVVGAD YKDDDDKNFP NNIQIGGLFP NQQSQEHAAF RFALSQLTEP PKLLPQIDIV NISDSFEMTY RFCSQFSKGV YAIFGFYERR TVNMLTSFCG ALHVCFITPS FPVDTSNQFV LQLRPELQEA LISIIDHYKW QTFVYIYDAD RGLSVLQRVL ...String:
MPYIFAFFCT GFLGAVVGAD YKDDDDKNFP NNIQIGGLFP NQQSQEHAAF RFALSQLTEP PKLLPQIDIV NISDSFEMTY RFCSQFSKGV YAIFGFYERR TVNMLTSFCG ALHVCFITPS FPVDTSNQFV LQLRPELQEA LISIIDHYKW QTFVYIYDAD RGLSVLQRVL DTAAEKNWQV TAVNILTTTE EGYRMLFQDL EKKKERLVVV DCESERLNAI LGQIVKLEKN GIGYHYILAN LGFMDIDLNK FKESGANVTG FQLVNYTDTI PARIMQQWRT SDSRDHTRVD WKRPKYTSAL TYDGVKVMAE AFQSLRRQRI DISRRGNAGD CLANPAVPWG QGIDIQRALQ QVRFEGLTGN VQFNEKGRRT NYTLHVIEMK HDGIRKIGYW NEDDKFVPAA TDAQAGGDNS SVQNRTYIVT TILEDPYVML KKNANQFEGN DRYEGYCVEL AAEIAKHVGY SYRLEIVSDG KYGARDPDTK AWNGMVGELV YGRADVAVAP LTITLVREEV IDFSKPFMSL GISIMIKKPQ KSKPGVFSFL DPLAYEIWMC IVFAYIGVSV VLFLVSRFSP YEWHSEEFEE GRDQTTSDQS NEFGIFNSLW FSLGAFMQQG CDISPRSLSG RIVGGVWWFF TLIIISSYTA NLAAFLTVER MVSPIESAED LAKQTEIAYG TLEAGSTKEF FRRSKIAVFE KMWTYMKSAE PSVFVRTTEE GMIRVRKSKG KYAYLLESTM NEYIEQRKPC DTMKVGGNLD SKGYGIATPK GSALRGPVNL AVLKLSEQGV LDKLKSKWWY DKGECGSKDS GSKDKTSALS LSNVAGVFYI LIGGLGLAML VALIEFCYKS RSESKRMKGF CLIPQQSINE AIRTSTLPRN SGAGASGGGG SGENGRVVSQ DFPKSMQSIP CMSHSSGMPL GATGL

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Macromolecule #2: AMPA Glutamate Receptor 2, isoform FLIP, Q/R edited

MacromoleculeName: AMPA Glutamate Receptor 2, isoform FLIP, Q/R edited / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MQKIMHISVL LSPVLWGLIF GVSSNSIQIG GLFPRGADQE YSAFRVGMVQ FSTSEFRLTP HIDNLEVANS FAVTNAFCSQ FSRGVYAIFG FYDKKSVNTI TSFCGTLHVS FITPSFPTDG THPFVIQMRP DLKGALLSLI EYYQWDKFAY LYDSDRGLST LQAVLDSAAE ...String:
MQKIMHISVL LSPVLWGLIF GVSSNSIQIG GLFPRGADQE YSAFRVGMVQ FSTSEFRLTP HIDNLEVANS FAVTNAFCSQ FSRGVYAIFG FYDKKSVNTI TSFCGTLHVS FITPSFPTDG THPFVIQMRP DLKGALLSLI EYYQWDKFAY LYDSDRGLST LQAVLDSAAE KKWQVTAINV GNINNDKKDE TYRSLFQDLE LKKERRVILD CERDKVNDIV DQVITIGKHV KGYHYIIANL GFTDGDLLKI QFGGANVSGF QIVDYDDSLV SKFIERWSTL EEKEYPGAHT ATIKYTSALT YDAVQVMTEA FRNLRKQRIE ISRRGNAGDC LANPAVPWGQ GVEIERALKQ VQVEGLSGNI KFDQNGKRIN YTINIMELKT NGPRKIGYWS EVDKMVVTLT ELPSGNDTSG LENKTVVVTT ILESPYVMMK KNHEMLEGNE RYEGYCVDLA AEIAKHCGFK YKLTIVGDGK YGARDADTKI WNGMVGELVY GKADIAIAPL TITLVREEVI DFSKPFMSLG ISIMIKKPQK SKPGVFSFLD PLAYEIWMCI VFAYIGVSVV LFLVSRFSPY EWHTEEFEDG RETQSSESTN EFGIFNSLWF SLGAFMRQGC DISPRSLSGR IVGGVWWFFT LIIISSYTAN LAAFLTVERM VSPIESAEDL SKQTEIAYGT LDSGSTKEFF RRSKIAVFDK MWTYMRSAEP SVFVRTTAEG VARVRKSKGK YAYLLESTMN EYIEQRKPCD TMKVGGNLDS KGYGIATPKG SSLGTPVNLA VLKLSEQGVL DKLKNKWWYD KGECGAKDSG SKEKTSALSL SNVAGVFYIL VGGLGLAMLV ALIEFCYKSR AEAKRMKVAK NPQNINPSSS

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Macromolecule #3: TARP gamma 8

MacromoleculeName: TARP gamma 8 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GESLKRWNEE RGLWCEKGVQ VLLTTIGAFA AFGLMTIAIS TDYWLYTRAL ICNTTNLTAG DDGPPHRGGS GSSEKKDPGG LTHSGLWRIC CLEGLKRGVC VKINHFPEDT DYDHDSAEYL LRVVRASSIF PILSAILLLL GGVCVAASRV YKSKRNIILG AGILFVAAGL ...String:
GESLKRWNEE RGLWCEKGVQ VLLTTIGAFA AFGLMTIAIS TDYWLYTRAL ICNTTNLTAG DDGPPHRGGS GSSEKKDPGG LTHSGLWRIC CLEGLKRGVC VKINHFPEDT DYDHDSAEYL LRVVRASSIF PILSAILLLL GGVCVAASRV YKSKRNIILG AGILFVAAGL SNIIGVIVYI SANAGEPGPK RDEEKKNHYS YGWSFYFGGL SFILAEVIGV LAVNIYIERS REAHCQSRSD LLKAGGGAGG SGGSGPSAIL RLPSYRFRYR RRSRSSSRGS SEASPSRDAS PGGPGGPGFA STDISMYTLS RDPSKGSVAA GLASAGGGGG GAGVGAYGGA AGAAGGGGTG SERDRGSSAG FLTLHNAFPK EAASGVTVTV TGPPAAPAPA PPAPAAPAPG TLSKEAAASN TNTLNRKLEV LFQ

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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

Concentration2.5 mg/mL
BufferpH: 8
Details: 25 mM TRIS 150 mM NaCl 0.02 % GDN 87 uM CTZ 100 mM L-Glu
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 3-4 second blots.
DetailsSample was incubated with 87 uM CTZ for 30 minutes and 100 mM L-Glutamate was added before grid preparation

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.34 µm / Nominal magnification: 81000
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: 9664 / Average exposure time: 4.0 sec. / Average electron dose: 51.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL / In silico model: Initial model was obtained in Relion
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.39 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1)
Details: In the final step of refinement a mask covering only the transmembrane region of the complex was created and refinement was performed. After that, postprocessed was performed in relion
Number images used: 83344
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7qhb


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsThis map was used for guiding manual model building of the TMD region in the PDB code 7QHB (map 13969), which also contains the LBD region
RefinementProtocol: RIGID BODY FIT

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