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- PDB-6x2l: hEAAT3-IFS-Na -

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Basic information

Entry
Database: PDB / ID: 6x2l
TitlehEAAT3-IFS-Na
ComponentsExcitatory amino acid transporter 3
KeywordsTRANSPORT PROTEIN / humant Excitatory amino acid transporter 3
Function / homology
Function and homology information


response to decreased oxygen levels / D-aspartate transmembrane transport / response to anesthetic / regulation of protein targeting to membrane / neurotransmitter receptor transport to plasma 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 ...response to decreased oxygen levels / D-aspartate transmembrane transport / response to anesthetic / regulation of protein targeting to membrane / neurotransmitter receptor transport to plasma 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 / monoatomic anion channel activity / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity / L-glutamate import / cellular response to mercury ion / Transport of inorganic cations/anions and amino acids/oligopeptides / conditioned place preference / zinc ion transmembrane transport / L-glutamate transmembrane transport / retina layer formation / cellular response to bisphenol A / L-glutamate transmembrane transporter activity / L-aspartate transmembrane transport / D-aspartate import across plasma membrane / cellular response to ammonium ion / proximal dendrite / glutathione biosynthetic process / righting reflex / L-aspartate transmembrane transporter activity / L-aspartate import across plasma membrane / grooming behavior / intracellular glutamate homeostasis / L-glutamate import across plasma membrane / Glutamate Neurotransmitter Release Cycle / apical dendrite / neurotransmitter transport / response to morphine / transepithelial transport / intracellular zinc ion homeostasis / glutamate receptor signaling pathway / cellular response to cocaine / chloride transmembrane transporter activity / blood vessel morphogenesis / motor neuron apoptotic process / motor behavior / glutamate binding / dopamine receptor signaling pathway / adult behavior / maintenance of blood-brain barrier / positive regulation of heart rate / dopamine metabolic process / postsynaptic modulation of chemical synaptic transmission / 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 / synaptic cleft / axon terminus / monoatomic ion transport / chloride transmembrane transport / response to amphetamine / neurogenesis / dendritic shaft / locomotory behavior / long-term synaptic potentiation / cell periphery / synapse organization / regulation of protein phosphorylation / brain development / Schaffer collateral - CA1 synapse / memory / cytokine-mediated signaling pathway / recycling endosome membrane / presynapse / late endosome membrane / cellular response to oxidative stress / gene expression / chemical synaptic transmission / perikaryon / early endosome membrane / negative regulation of neuron apoptotic process / dendritic spine / response to xenobiotic stimulus / membrane raft / apical plasma membrane / axon / dendrite / neuronal cell body / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.85 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 20, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 17, 2021Provider: 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

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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


Theoretical massNumber of molelcules
Total (without water)171,8263
Polymers171,8263
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, homolog, microscopy
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: 57275.168 Da / Num. of mol.: 3 / Mutation: N178T, N195T
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

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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

Component
IDNameTypeEntity IDParent-IDSource
1inward facing hEAAT3 trimerCOMPLEXall0RECOMBINANT
2inward facing hEAAT3 trimerCOMPLEXall1RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
2200 mMSodium ChlorideNaClSodium chloride1
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 FIELDBright-field microscopy / 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
10RELION3initial Euler assignment
11RELION3final Euler assignment
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1717667
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 554920 / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0099534
ELECTRON MICROSCOPYf_angle_d0.9712936
ELECTRON MICROSCOPYf_dihedral_angle_d18.1211293
ELECTRON MICROSCOPYf_chiral_restr0.0551644
ELECTRON MICROSCOPYf_plane_restr0.0071572

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