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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


D-aspartate transmembrane transport / D-aspartate transmembrane transporter activity / Defective SLC1A1 is implicated in schizophrenia 18 (SCZD18) and dicarboxylic aminoaciduria (DCBXA) / SLC-mediated transport of amino acids / distal dendrite / L-cysteine transport / L-cysteine transmembrane transporter activity / L-glutamate import / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity ...D-aspartate transmembrane transport / D-aspartate transmembrane transporter activity / Defective SLC1A1 is implicated in schizophrenia 18 (SCZD18) and dicarboxylic aminoaciduria (DCBXA) / SLC-mediated transport of amino acids / distal dendrite / L-cysteine transport / L-cysteine transmembrane transporter activity / L-glutamate import / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity / L-glutamate transmembrane transporter activity / glutathione biosynthetic process / L-glutamate transmembrane transport / L-aspartate transmembrane transport / grooming behavior / zinc ion transmembrane transport / L-aspartate transmembrane transporter activity / Glutamate Neurotransmitter Release Cycle / L-aspartate import across plasma membrane / D-aspartate import across plasma membrane / monoatomic anion channel activity / proximal dendrite / blood vessel morphogenesis / L-glutamate import across plasma membrane / intracellular zinc ion homeostasis / transepithelial transport / apical dendrite / cellular response to cocaine / chloride transmembrane transporter activity / G protein-coupled dopamine receptor signaling pathway / superoxide metabolic process / glutamate receptor signaling pathway / adult behavior / response to morphine / perisynaptic space / maintenance of blood-brain barrier / dopamine metabolic process / motor behavior / neurotransmitter transport / amino acid transport / asymmetric synapse / glial cell projection / conditioned place preference / postsynaptic modulation of chemical synaptic transmission / positive regulation of heart rate / transport across blood-brain barrier / synaptic cleft / neurogenesis / axon terminus / response to amphetamine / chloride transmembrane transport / cell periphery / dendritic shaft / locomotory behavior / brain development / synapse organization / recycling endosome / Schaffer collateral - CA1 synapse / recycling endosome membrane / memory / cytokine-mediated signaling pathway / apical part of cell / late endosome membrane / neuron apoptotic process / presynapse / retina development in camera-type eye / gene expression / early endosome membrane / chemical synaptic transmission / negative regulation of neuron apoptotic process / dendritic spine / perikaryon / apical plasma membrane / membrane raft / axon / neuronal cell body / dendrite / synapse / cell surface / extracellular exosome / membrane / metal ion binding / identical protein 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-
UpdateMay 21, 2025-
Current statusMay 21, 2025Processing 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
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 288 pix.
= 239.616 Å		0.83 Å/pix.
x 288 pix.
= 239.616 Å		0.83 Å/pix.
x 288 pix.
= 239.616 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.832 Å
Density

Histogram

Histogram (log scale)
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
CTF correctionSoftware - Name: RELION (ver. 3.0) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
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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