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- PDB-8ctc: Human excitatory amino acid transporter 3 (EAAT3) with bound glut... -

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Basic information

Entry
Database: PDB / ID: 8ctc
TitleHuman excitatory amino acid transporter 3 (EAAT3) with bound glutamate in an intermediate outward facing state
ComponentsExcitatory amino acid transporter 3
KeywordsTRANSPORT PROTEIN
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 / L-glutamate import / response to decreased oxygen levels / cellular response to mercury ion / : / L-glutamate transmembrane transporter activity / retina layer formation / L-glutamate transmembrane transport / glutathione biosynthetic process / D-aspartate import across plasma membrane / L-aspartate transmembrane transport / cellular response to ammonium ion / righting reflex / zinc ion transmembrane transport / L-aspartate transmembrane transporter activity / cellular response to bisphenol A / intracellular glutamate homeostasis / grooming behavior / L-aspartate import across plasma membrane / Glutamate Neurotransmitter Release Cycle / monoatomic anion channel activity / L-glutamate import across plasma membrane / proximal dendrite / transepithelial transport / apical dendrite / intracellular zinc ion homeostasis / blood vessel morphogenesis / cellular response to cocaine / chloride transmembrane transporter activity / motor neuron apoptotic process / G protein-coupled dopamine receptor signaling pathway / response to anesthetic / response to morphine / glutamate receptor signaling pathway / superoxide metabolic process / neurotransmitter transport / heart contraction / perisynaptic space / maintenance of blood-brain barrier / dopamine metabolic process / adult behavior / motor behavior / asymmetric synapse / conditioned place preference / glial cell projection / synaptic cleft / behavioral fear response / postsynaptic modulation of chemical synaptic transmission / response to axon injury / positive regulation of heart rate / transport across blood-brain barrier / monoatomic ion transport / neurogenesis / axon terminus / chloride transmembrane transport / response to amphetamine / dendritic shaft / cell periphery / locomotory behavior / synapse organization / brain development / Schaffer collateral - CA1 synapse / memory / long-term synaptic potentiation / recycling endosome membrane / cytokine-mediated signaling pathway / late endosome membrane / presynapse / cellular response to oxidative stress / early endosome membrane / perikaryon / chemical synaptic transmission / gene expression / dendritic spine / negative regulation of neuron apoptotic process / apical plasma membrane / membrane raft / response to xenobiotic stimulus / axon / neuronal cell body / dendrite / cell surface / 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
GLUTAMIC ACID / : / Excitatory amino acid transporter 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsQiu, B. / Boudker, O.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R37NS085318 United States
CitationJournal: Nat Commun / Year: 2023
Title: Symport and antiport mechanisms of human glutamate transporters.
Authors: Biao Qiu / Olga Boudker /
Abstract: Excitatory amino acid transporters (EAATs) uptake glutamate into glial cells and neurons. EAATs achieve million-fold transmitter gradients by symporting it with three sodium ions and a proton, and ...Excitatory amino acid transporters (EAATs) uptake glutamate into glial cells and neurons. EAATs achieve million-fold transmitter gradients by symporting it with three sodium ions and a proton, and countertransporting a potassium ion via an elevator mechanism. Despite the availability of structures, the symport and antiport mechanisms still need to be clarified. We report high-resolution cryo-EM structures of human EAAT3 bound to the neurotransmitter glutamate with symported ions, potassium ions, sodium ions alone, or without ligands. We show that an evolutionarily conserved occluded translocation intermediate has a dramatically higher affinity for the neurotransmitter and the countertransported potassium ion than outward- or inward-facing transporters and plays a crucial role in ion coupling. We propose a comprehensive ion coupling mechanism involving a choreographed interplay between bound solutes, conformations of conserved amino acid motifs, and movements of the gating hairpin and the substrate-binding domain.
History
DepositionMay 13, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 10, 2023Provider: repository / Type: Initial release
Revision 1.1May 17, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jun 12, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / em_3d_fitting_list
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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)172,61318
Polymers171,3633
Non-polymers1,25015
Water21612
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: 57120.863 Da / Num. of mol.: 3
Mutation: C9A, C100A, C158A, N178T, N195T, C219A, C256W, K269C, W441C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC1A1, EAAC1, EAAT3 / Production host: Homo sapiens (human) / References: UniProt: P43005
#2: Chemical ChemComp-GLU / GLUTAMIC ACID


Type: L-peptide linking / Mass: 147.129 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C5H9NO4 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: Na / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-HG / MERCURY (II) ION


Mass: 200.590 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Hg / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 12 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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
1Human EAAT3 with glutamate boundCOMPLEX#10RECOMBINANT
2Human EAAT3 with glutamate boundCOMPLEX#11RECOMBINANT
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: 7.4 / Details: Tris is used for adjusting pH
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHepesC8H18N2O4S1
2200 mMsodium chlorideNaCl1
30.086 mMDigitonin glyco diosgeninC56H92O251
420 mML-glutamateC5H9NO41
SpecimenConc.: 4.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Human EAAT3 with glutamate bound
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: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50.266 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: UCSF ChimeraX / Version: 1.2/v9 / Classification: model building / URL: https://www.rbvi.ucsf.edu/chimerax/ / Os: macOS / Type: package
EM software
IDNameCategory
2Leginonimage acquisition
4CTFFINDCTF correction
7UCSF ChimeraXmodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13Cootmodel refinement
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 8511485
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 496972 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16X2Z

6x2z

16X2Z1PDBexperimental model
26X2L

6x2l

16X2L2PDBexperimental model

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