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- EMDB-22020: hEAAT3-Asymmetric-1o2i -

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Basic information

Entry
Database: EMDB / ID: EMD-22020
TitlehEAAT3-Asymmetric-1o2i
Map dataAsymmetric, Outward-facing bound, Inward-facing open
Sample
  • Complex: Asymmetric hEAAT3 trimer
    • Protein or peptide: Excitatory amino acid transporter 3
  • Ligand: ASPARTIC ACID
  • Ligand: SODIUM ION
KeywordsAsymmetric / Outward-facing bound / Inward-facing open / TRANSPORT PROTEIN
Function / homology
Function and homology information


response to decreased oxygen levels / D-aspartate transmembrane transport / response to anesthetic / regulation of protein targeting to membrane / D-aspartate transmembrane transporter activity / Defective SLC1A1 is implicated in schizophrenia 18 (SCZD18) and dicarboxylic aminoaciduria (DCBXA) / distal dendrite / cysteine transmembrane transporter activity / cysteine transport / high-affinity L-glutamate transmembrane transporter activity ...response to decreased oxygen levels / D-aspartate transmembrane transport / response to anesthetic / regulation of protein targeting to membrane / D-aspartate transmembrane transporter activity / Defective SLC1A1 is implicated in schizophrenia 18 (SCZD18) and dicarboxylic aminoaciduria (DCBXA) / distal dendrite / cysteine transmembrane transporter activity / cysteine transport / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity / L-glutamate import / neurotransmitter receptor transport to plasma membrane / cellular response to mercury ion / Transport of inorganic cations/anions and amino acids/oligopeptides / zinc ion transmembrane transport / retina layer formation / L-glutamate transmembrane transport / L-glutamate transmembrane transporter activity / monoatomic anion channel activity / cellular response to bisphenol A / L-aspartate transmembrane transport / D-aspartate import across plasma membrane / cellular response to ammonium ion / proximal dendrite / righting reflex / L-aspartate transmembrane transporter activity / glutathione biosynthetic process / L-aspartate import across plasma membrane / grooming behavior / intracellular glutamate homeostasis / Glutamate Neurotransmitter Release Cycle / L-glutamate import across plasma membrane / conditioned place preference / transepithelial transport / apical dendrite / intracellular zinc ion homeostasis / glutamate receptor signaling pathway / response to morphine / neurotransmitter transport / cellular response to cocaine / blood vessel morphogenesis / motor behavior / motor neuron apoptotic process / glutamate binding / chloride transmembrane transporter activity / G protein-coupled dopamine receptor signaling pathway / adult behavior / positive regulation of heart rate / postsynaptic modulation of chemical synaptic transmission / maintenance of blood-brain barrier / dopamine metabolic process / superoxide metabolic process / heart contraction / perisynaptic space / glial cell projection / transport across blood-brain barrier / cellular response to organic cyclic compound / asymmetric synapse / response to axon injury / behavioral fear response / monoatomic ion transport / synaptic cleft / axon terminus / chloride transmembrane transport / response to amphetamine / neurogenesis / dendritic shaft / locomotory behavior / cell periphery / long-term synaptic potentiation / synapse organization / regulation of protein phosphorylation / Schaffer collateral - CA1 synapse / brain development / memory / cytokine-mediated signaling pathway / recycling endosome membrane / presynapse / late endosome membrane / gene expression / cellular response to oxidative stress / early endosome membrane / chemical synaptic transmission / perikaryon / negative regulation of neuron apoptotic process / dendritic spine / response to xenobiotic stimulus / membrane raft / apical plasma membrane / axon / neuronal cell body / dendrite / cell surface / endoplasmic reticulum / extracellular exosome / membrane / identical protein binding / 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 3
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.42 Å
AuthorsQiu B / Matthies D
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS064357 United States
CitationJournal: Sci Adv / Year: 2021
Title: Cryo-EM structures of excitatory amino acid transporter 3 visualize coupled substrate, sodium, and proton binding and transport.
Authors: Biao Qiu / Doreen Matthies / Eva Fortea / Zhiheng Yu / Olga Boudker /
Abstract: Human excitatory amino acid transporter 3 (hEAAT3) mediates glutamate uptake in neurons, intestine, and kidney. Here, we report cryo-EM structures of hEAAT3 in several functional states where the ...Human excitatory amino acid transporter 3 (hEAAT3) mediates glutamate uptake in neurons, intestine, and kidney. Here, we report cryo-EM structures of hEAAT3 in several functional states where the transporter is empty, bound to coupled sodium ions only, or fully loaded with three sodium ions, a proton, and the substrate aspartate. The structures suggest that hEAAT3 operates by an elevator mechanism involving three functionally independent subunits. When the substrate-binding site is near the cytoplasm, it has a remarkably low affinity for the substrate, perhaps facilitating its release and allowing the rapid transport turnover. The mechanism of the coupled uptake of the sodium ions and the substrate is conserved across evolutionarily distant families and is augmented by coupling to protons in EAATs. The structures further suggest a mechanism by which a conserved glutamate residue mediates proton symport.
History
DepositionMay 21, 2020-
Header (metadata) releaseMar 17, 2021-
Map releaseMar 17, 2021-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0185
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0185
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6x3e
  • Surface level: 0.0185
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22020.png

Downloads & links

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Map

FileDownload / File: emd_22020.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAsymmetric, Outward-facing bound, Inward-facing open
Voxel sizeX=Y=Z: 0.832 Å
Density
Contour LevelBy AUTHOR: 0.0185 / Movie #1: 0.0185
Minimum - Maximum-0.05292642 - 0.100022614
Average (Standard dev.)0.00033235407 (±0.003055578)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 239.616 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8320.8320.832
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z239.616239.616239.616
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-0.0530.1000.000

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Supplemental data

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Sample components

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Entire : Asymmetric hEAAT3 trimer

EntireName: Asymmetric hEAAT3 trimer
Components
  • Complex: Asymmetric hEAAT3 trimer
    • Protein or peptide: Excitatory amino acid transporter 3
  • Ligand: ASPARTIC ACID
  • Ligand: SODIUM ION

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Supramolecule #1: Asymmetric hEAAT3 trimer

SupramoleculeName: Asymmetric hEAAT3 trimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Excitatory amino acid transporter 3

MacromoleculeName: Excitatory amino acid transporter 3 / type: protein_or_peptide / ID: 1
Details: The Glycine and Proline at the N terminal are the residues left after PreScission Protease treatment
Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 57.301168 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GPMGKPARKG CEWKRFLKNN WVLLSTVAAV VLGITTGVLV REHSNLSTLE KFYFAFPGEI LMRMLKLIIL PLIISSMITG VAALDSNVS GKIGLRAVVY YFCTTLIAVI LGIVLVVSIK PGVTQKVGEI ARTGSTPEVS TVDAMLDLIR NMFPENLVQA C FQQYKTKR ...String:
GPMGKPARKG CEWKRFLKNN WVLLSTVAAV VLGITTGVLV REHSNLSTLE KFYFAFPGEI LMRMLKLIIL PLIISSMITG VAALDSNVS GKIGLRAVVY YFCTTLIAVI LGIVLVVSIK PGVTQKVGEI ARTGSTPEVS TVDAMLDLIR NMFPENLVQA C FQQYKTKR EEVKPPSDPE MNMTEESFTA VMTTAISKNK TKEYKIVGMY SDGINVLGLI VFCLVFGLVI GKMGEKGQIL VD FFNALSD ATMKIVQIIM CYMPLGILFL IAGKIIEVED WEIFRKLGLY MATVLTGLAI HSIVILPLIY FIVVRKNPFR FAM GMAQAL LTALMISSSS ATLPVTFRCA EENNQVDKRI TRFVLPVGAT INMDGTALYE AVAAVFIAQL NDLDLGIGQI ITIS ITATS ASIGAAGVPQ AGLVTMVIVL SAVGLPAEDV TLIIAVDWLL DRFRTMVNVL GDAFGTGIVE KLSKKELEQM DVSSE VNIV NPFALESTIL DNEDSDTKKS YVNGGFAVDK SDTISFTQTS QF

UniProtKB: Excitatory amino acid transporter 3

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Macromolecule #2: ASPARTIC ACID

MacromoleculeName: ASPARTIC ACID / type: ligand / ID: 2 / Number of copies: 1 / Formula: ASP
Molecular weightTheoretical: 133.103 Da
Chemical component information

ChemComp-ASP:
ASPARTIC ACID

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Macromolecule #3: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 3 / Number of copies: 3
Molecular weightTheoretical: 22.99 Da

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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

Concentration6 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris
200.0 mMNaClsodium chloride
1.0 mMC4H7NO4L-Aspartate
1.0 mMC9H15O6PTCEP
0.086 mMC56H92O25Digitonin glyco diosgenin
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot 3s.
DetailsThis sample was mono disperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 190599
Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.42 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 158349
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 5
FSC plot (resolution estimation)

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