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- PDB-6x3f: hEAAT3-IFS-Apo -

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Basic information

Entry
Database: PDB / ID: 6x3f
TitlehEAAT3-IFS-Apo
ComponentsExcitatory amino acid transporter 3
KeywordsTRANSPORT PROTEIN / hEAAT3 inward-facing Apo state
Function / homology
Function and homology information


D-aspartate transmembrane transport / 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 transport / cysteine transmembrane transporter activity / neurotransmitter receptor transport to plasma membrane / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity ...D-aspartate transmembrane transport / 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 transport / cysteine transmembrane transporter activity / neurotransmitter receptor transport to plasma membrane / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity / response to decreased oxygen levels / L-glutamate import / cellular response to mercury ion / Transport of inorganic cations/anions and amino acids/oligopeptides / retina layer formation / L-glutamate transmembrane transporter activity / cellular response to ammonium ion / L-glutamate transmembrane transport / glutathione biosynthetic process / D-aspartate import across plasma membrane / L-aspartate transmembrane transport / righting reflex / zinc ion transmembrane transport / cellular response to bisphenol A / L-aspartate transmembrane transporter activity / grooming behavior / intracellular glutamate homeostasis / L-aspartate import across plasma membrane / Glutamate Neurotransmitter Release Cycle / proximal dendrite / monoatomic anion channel activity / L-glutamate import across plasma membrane / apical dendrite / transepithelial transport / response to anesthetic / conditioned place preference / intracellular zinc ion homeostasis / cellular response to cocaine / chloride transmembrane transporter activity / blood vessel morphogenesis / motor neuron apoptotic process / response to morphine / G protein-coupled dopamine receptor signaling pathway / motor behavior / glutamate receptor signaling pathway / glutamate binding / neurotransmitter transport / superoxide metabolic process / heart contraction / maintenance of blood-brain barrier / perisynaptic space / dopamine metabolic process / adult behavior / : / asymmetric synapse / glial cell projection / response to axon injury / behavioral fear response / synaptic cleft / postsynaptic modulation of chemical synaptic transmission / transport across blood-brain barrier / positive regulation of heart rate / monoatomic ion transport / neurogenesis / axon terminus / response to amphetamine / chloride transmembrane transport / dendritic shaft / cell periphery / locomotory behavior / synapse organization / cytokine-mediated signaling pathway / brain development / Schaffer collateral - CA1 synapse / memory / long-term synaptic potentiation / recycling endosome membrane / late endosome membrane / presynapse / cellular response to oxidative stress / early endosome membrane / gene expression / chemical synaptic transmission / dendritic spine / perikaryon / negative regulation of neuron apoptotic process / apical plasma membrane / membrane raft / response to xenobiotic stimulus / axon / external side of plasma membrane / neuronal cell body / dendrite / endoplasmic reticulum / extracellular exosome / metal ion binding / identical protein binding / membrane / 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
CHOLINE ION / Excitatory amino acid transporter 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.03 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 28, 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 28, 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)172,2166
Polymers171,9043
Non-polymers3133
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, EM
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-CHT / CHOLINE ION


Mass: 104.171 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C5H14NO / 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: inward facing hEAAT3 trimer Apo state / 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 mMTris-baseC4H11NO31
2100 mMcholine chlorideC5H14ClNO1
31 mMTCEPC9H15O6P1
40.086 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: GOLD / Grid mesh size: 300 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
12RELION3classification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1918723
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.03 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 435398 / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0069501
ELECTRON MICROSCOPYf_angle_d0.93912894
ELECTRON MICROSCOPYf_dihedral_angle_d20.5181293
ELECTRON MICROSCOPYf_chiral_restr0.0581638
ELECTRON MICROSCOPYf_plane_restr0.0081563

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