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- PDB-6x3e: hEAAT3-Asymmetric-1o2i -

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Basic information

Entry
Database: PDB / ID: 6x3e
TitlehEAAT3-Asymmetric-1o2i
ComponentsExcitatory amino acid transporter 3
KeywordsTRANSPORT PROTEIN / Asymmetric / Outward-facing bound / Inward-facing open
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
Proton glutamate symport protein / Sodium:dicarboxylate symporter / : / 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. / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
ASPARTIC ACID / Excitatory amino acid transporter 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.42 Å
AuthorsQiu, B. / Matthies, D. / Boudker, O.
Funding support United States, 1items
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, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 17, 2021Provider: repository / Type: Initial release
Revision 1.0Mar 17, 2021Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 17, 2021Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 17, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 17, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Mar 17, 2021Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 17, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 17, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2May 21, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details
Item: _em_admin.last_update / _em_software.name / _pdbx_entry_details.has_protein_modification
Revision 1.1May 21, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

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Assembly

Deposited unit
A: Excitatory amino acid transporter 3
B: Excitatory amino acid transporter 3
C: Excitatory amino acid transporter 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)172,1067
Polymers171,9043
Non-polymers2024
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Excitatory amino acid transporter 3 / Excitatory amino-acid carrier 1 / Neuronal and epithelial glutamate transporter / Sodium-dependent ...Excitatory amino-acid carrier 1 / Neuronal and epithelial glutamate transporter / Sodium-dependent glutamate/aspartate transporter 3 / Solute carrier family 1 member 1


Mass: 57301.168 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: The Glycine and Proline at the N terminal are the residues left after PreScission Protease treatment
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC1A1, EAAC1, EAAT3 / Production host: Homo sapiens (human) / References: UniProt: P43005
#2: Chemical ChemComp-ASP / ASPARTIC ACID


Type: L-peptide linking / Mass: 133.103 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H7NO4 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Na / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Asymmetric hEAAT3 trimer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
2200 mMsodium chlorideNaCl1
31 mML-AspartateC4H7NO41
41 mMTCEPC9H15O6P1
50.086 mMDigitonin glyco diosgeninC56H92O251
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was mono disperse
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot 3s

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18rc1_3769: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3particle selection
2SerialEMimage acquisition
4RELION3CTF correction
8PHENIXmodel refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 190599
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.42 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 158349 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0069603
ELECTRON MICROSCOPYf_angle_d0.91913028
ELECTRON MICROSCOPYf_dihedral_angle_d18.7481302
ELECTRON MICROSCOPYf_chiral_restr0.0551654
ELECTRON MICROSCOPYf_plane_restr0.0071585

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