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- EMDB-12524: Cryo-EM structure of Human excitatory amino acid transporters-1 (... -

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Basic information

Entry
Database: EMDB / ID: EMD-12524
TitleCryo-EM structure of Human excitatory amino acid transporters-1 (EAAT1) in potassium buffer
Map dataCryo-EM map of Human excitatory amino acid transporters (EAATs)
Sample
  • Complex: Human excitatory amino acid transporter 1
    • Protein or peptide: Excitatory amino acid transporter 1
KeywordsHuman Membrane Protein / Transporter / Glutamate transporter / MEMBRANE PROTEIN
Function / homology
Function and homology information


Defective SLC1A3 causes episodic ataxia 6 (EA6) / auditory behavior / Astrocytic Glutamate-Glutamine Uptake And Metabolism / membrane protein complex / neurotransmitter uptake / cranial nerve development / cell morphogenesis involved in neuron differentiation / gamma-aminobutyric acid biosynthetic process / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity ...Defective SLC1A3 causes episodic ataxia 6 (EA6) / auditory behavior / Astrocytic Glutamate-Glutamine Uptake And Metabolism / membrane protein complex / neurotransmitter uptake / cranial nerve development / cell morphogenesis involved in neuron differentiation / gamma-aminobutyric acid biosynthetic process / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity / L-glutamate import / Transport of inorganic cations/anions and amino acids/oligopeptides / L-glutamate transmembrane transport / L-glutamate transmembrane transporter activity / D-aspartate import across plasma membrane / L-aspartate import across plasma membrane / neutral L-amino acid transmembrane transporter activity / Glutamate Neurotransmitter Release Cycle / L-glutamate import across plasma membrane / transepithelial transport / intracellular sodium ion homeostasis / neurotransmitter transport / cellular response to cocaine / glutamate binding / neuromuscular process controlling balance / transport across blood-brain barrier / response to light stimulus / positive regulation of synaptic transmission / monoatomic ion transport / potassium ion transmembrane transport / chloride transmembrane transport / basal plasma membrane / sensory perception of sound / response to wounding / cytoplasmic vesicle / chemical synaptic transmission / response to xenobiotic stimulus / neuron projection / response to antibiotic / neuronal cell body / synapse / perinuclear region of cytoplasm / cell surface / membrane / metal ion binding / plasma membrane
Similarity search - Function
Sodium:dicarboxylate symporter family signature 2. / Sodium:dicarboxylate symporter / Sodium:dicarboxylate symporter, conserved site / Sodium:dicarboxylate symporter superfamily / Sodium:dicarboxylate symporter family / Sodium:dicarboxylate symporter family signature 1.
Similarity search - Domain/homology
Excitatory amino acid transporter 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.99 Å
AuthorsKumar A / Reyes N
Funding support France, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)309657 France
CitationJournal: EMBO J / Year: 2022
Title: The ion-coupling mechanism of human excitatory amino acid transporters.
Authors: Juan C Canul-Tec / Anand Kumar / Jonathan Dhenin / Reda Assal / Pierre Legrand / Martial Rey / Julia Chamot-Rooke / Nicolas Reyes /
Abstract: Excitatory amino acid transporters (EAATs) maintain glutamate gradients in the brain essential for neurotransmission and to prevent neuronal death. They use ionic gradients as energy source and co- ...Excitatory amino acid transporters (EAATs) maintain glutamate gradients in the brain essential for neurotransmission and to prevent neuronal death. They use ionic gradients as energy source and co-transport transmitter into the cytoplasm with Na and H , while counter-transporting K to re-initiate the transport cycle. However, the molecular mechanisms underlying ion-coupled transport remain incompletely understood. Here, we present 3D X-ray crystallographic and cryo-EM structures, as well as thermodynamic analysis of human EAAT1 in different ion bound conformations, including elusive counter-transport ion bound states. Binding energies of Na and H , and unexpectedly Ca , are coupled to neurotransmitter binding. Ca competes for a conserved Na site, suggesting a regulatory role for Ca in glutamate transport at the synapse, while H binds to a conserved glutamate residue stabilizing substrate occlusion. The counter-transported ion binding site overlaps with that of glutamate, revealing the K -based mechanism to exclude the transmitter during the transport cycle and to prevent its neurotoxic release on the extracellular side.
History
DepositionFeb 28, 2021-
Header (metadata) releaseOct 13, 2021-
Map releaseOct 13, 2021-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7npw
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12524.png

Downloads & links

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Map

FileDownload / File: emd_12524.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of Human excitatory amino acid transporters (EAATs)
Voxel sizeX=Y=Z: 0.814 Å
Density
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.1
Minimum - Maximum-0.2050666 - 0.39906365
Average (Standard dev.)0.0005771564 (±0.012244673)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 325.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8140.8140.814
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z325.600325.600325.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.2050.3990.001

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Supplemental data

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Sample components

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Entire : Human excitatory amino acid transporter 1

EntireName: Human excitatory amino acid transporter 1
Components
  • Complex: Human excitatory amino acid transporter 1
    • Protein or peptide: Excitatory amino acid transporter 1

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Supramolecule #1: Human excitatory amino acid transporter 1

SupramoleculeName: Human excitatory amino acid transporter 1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Excitatory amino acid transporter 1

MacromoleculeName: Excitatory amino acid transporter 1 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 51.078273 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: AKKKVQNITK EDVKSYLFRN AFVLLTVTAV IVGTILGFTL RPYRMSYREV KYFSFPGELL MRMLQMLVLP LIISSLVTGM AALDSKASG KMGMRAVVYY MTTTIIAVVI GIIIVIIIHP GKGTKENMHR EGKIVRVTAA DAFLDLIRNM FPPNLVEACF K QFKTNYEK ...String:
AKKKVQNITK EDVKSYLFRN AFVLLTVTAV IVGTILGFTL RPYRMSYREV KYFSFPGELL MRMLQMLVLP LIISSLVTGM AALDSKASG KMGMRAVVYY MTTTIIAVVI GIIIVIIIHP GKGTKENMHR EGKIVRVTAA DAFLDLIRNM FPPNLVEACF K QFKTNYEK RSFKVPIQAN ETLVGAVINN VSEAMETLTR ITEELVPVPG SVNGVNALGL VVFSMCFGFV IGNMKEQGQA LR EFFDSLN EAIMRLVAVI MWYAPVGILF LIAGKIVEME DMGVIGGQLA MYTVTVIVGL LIHAVIVLPL LYFLVTRKNP WVF IGGLLQ ALITALGTSS SSATLPITFK CLEENNGVDK RVTRFVLPVG ATINMDGTAL YEALAAIFIA QVNNFELNFG QIIT ISITA TAASIGAAGI PQAGLVTMVI VLTSVGLPTD DITLIIAVDW FLDRLRTTTN VLGDSLGAGI VEHL

UniProtKB: Excitatory amino acid transporter 1

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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: 7.4
Component:
ConcentrationFormulaName
50.0 mMHEPES2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
100.0 mMKClPotassium Chloride
1.0 mMTCEPtris(2-carboxyethyl)phosphine
0.0084 PercentGDNGDN101
0.0017 PercentCHSCholesterol Hemisuccinate
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: Grids were blotted for 4 Sec.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 13607 / Average electron dose: 42.3 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2298481
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6gct


Details: We used our crystal structure and ASCT2 PDB from the database (6GCT)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.99 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 34433
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC

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Atomic model buiding 1

DetailsFor model building, the scaD and tranD of Rb+/Ba2+ bound X-ray structure were fitted into the EM map as separate rigid-bodies using Chimera.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7npw:
Cryo-EM structure of Human excitatory amino acid transporters-1 (EAAT1) in potassium buffer

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