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- PDB-7npw: Cryo-EM structure of Human excitatory amino acid transporters-1 (... -

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Basic information

Entry
Database: PDB / ID: 7npw
TitleCryo-EM structure of Human excitatory amino acid transporters-1 (EAAT1) in potassium buffer
ComponentsExcitatory amino acid transporter 1
KeywordsMEMBRANE PROTEIN / Human Membrane Protein / Transporter / Glutamate transporter
Function / homology
Function and homology information


Defective SLC1A3 causes episodic ataxia 6 (EA6) / auditory behavior / Astrocytic Glutamate-Glutamine Uptake And Metabolism / membrane protein complex / neurotransmitter uptake / cranial nerve development / cell morphogenesis involved in neuron differentiation / gamma-aminobutyric acid biosynthetic process / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity ...Defective SLC1A3 causes episodic ataxia 6 (EA6) / auditory behavior / Astrocytic Glutamate-Glutamine Uptake And Metabolism / membrane protein complex / neurotransmitter uptake / cranial nerve development / cell morphogenesis involved in neuron differentiation / gamma-aminobutyric acid biosynthetic process / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity / L-glutamate import / Transport of inorganic cations/anions and amino acids/oligopeptides / L-glutamate transmembrane transport / L-glutamate transmembrane transporter activity / D-aspartate import across plasma membrane / L-aspartate import across plasma membrane / neutral L-amino acid transmembrane transporter activity / Glutamate Neurotransmitter Release Cycle / L-glutamate import across plasma membrane / transepithelial transport / intracellular sodium ion homeostasis / neurotransmitter transport / cellular response to cocaine / glutamate binding / neuromuscular process controlling balance / transport across blood-brain barrier / response to light stimulus / positive regulation of synaptic transmission / monoatomic ion transport / potassium ion transmembrane transport / chloride transmembrane transport / basal plasma membrane / sensory perception of sound / response to wounding / cytoplasmic vesicle / chemical synaptic transmission / response to xenobiotic stimulus / neuron projection / response to antibiotic / neuronal cell body / synapse / perinuclear region of cytoplasm / cell surface / membrane / 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 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.99 Å
AuthorsKumar, A. / Reyes, N.
Funding support France, 1items
OrganizationGrant numberCountry
European Research Council (ERC)309657 France
CitationJournal: EMBO J / Year: 2022
Title: The ion-coupling mechanism of human excitatory amino acid transporters.
Authors: Juan C Canul-Tec / Anand Kumar / Jonathan Dhenin / Reda Assal / Pierre Legrand / Martial Rey / Julia Chamot-Rooke / Nicolas Reyes /
Abstract: Excitatory amino acid transporters (EAATs) maintain glutamate gradients in the brain essential for neurotransmission and to prevent neuronal death. They use ionic gradients as energy source and co- ...Excitatory amino acid transporters (EAATs) maintain glutamate gradients in the brain essential for neurotransmission and to prevent neuronal death. They use ionic gradients as energy source and co-transport transmitter into the cytoplasm with Na and H , while counter-transporting K to re-initiate the transport cycle. However, the molecular mechanisms underlying ion-coupled transport remain incompletely understood. Here, we present 3D X-ray crystallographic and cryo-EM structures, as well as thermodynamic analysis of human EAAT1 in different ion bound conformations, including elusive counter-transport ion bound states. Binding energies of Na and H , and unexpectedly Ca , are coupled to neurotransmitter binding. Ca competes for a conserved Na site, suggesting a regulatory role for Ca in glutamate transport at the synapse, while H binds to a conserved glutamate residue stabilizing substrate occlusion. The counter-transported ion binding site overlaps with that of glutamate, revealing the K -based mechanism to exclude the transmitter during the transport cycle and to prevent its neurotoxic release on the extracellular side.
History
DepositionFeb 28, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 13, 2021Provider: repository / Type: Initial release
Revision 1.1Dec 1, 2021Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jan 12, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID
Revision 1.3Jan 19, 2022Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.4Jul 10, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
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  • Imaged by UCSF Chimera
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Assembly

Deposited unit
A: Excitatory amino acid transporter 1
B: Excitatory amino acid transporter 1
C: Excitatory amino acid transporter 1


Theoretical massNumber of molelcules
Total (without water)153,2353
Polymers153,2353
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, microscopy, Negative staining and cryoEM
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area7340 Å2
ΔGint-73 kcal/mol
Surface area62470 Å2
MethodPISA

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Components

#1: Protein Excitatory amino acid transporter 1 / Sodium-dependent glutamate/aspartate transporter 1 / GLAST-1 / Solute carrier family 1 member 3


Mass: 51078.273 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC1A3, EAAT1, GLAST, GLAST1 / Plasmid: pcDNA3.1 + / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: P43003

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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: Human excitatory amino acid transporter 1 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK-293F / Plasmid: pcDNA3.1
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
150 mM2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acidHEPES1
2100 mMPotassium ChlorideKCl1
31 mMtris(2-carboxyethyl)phosphineTCEP1
40.0084 PercentGDN101GDN1
50.0017 PercentCholesterol HemisuccinateCHS1
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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: 277.15 K / Details: Grids were blotted for 4 Sec

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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 defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 42.3 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 13607
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4GctfCTF correction
7UCSF Chimera1.13.1model fitting
8Coot0.8.9.2model fitting
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
14PHENIX1.18.2model refinement
CTF correctionDetails: Gctf / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2298481
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.99 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 34433 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: For model building, the scaD and tranD of Rb+/Ba2+ bound X-ray structure were fitted into the EM map as separate rigid-bodies using Chimera.
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0029885
ELECTRON MICROSCOPYf_angle_d0.64413428
ELECTRON MICROSCOPYf_dihedral_angle_d21.2271344
ELECTRON MICROSCOPYf_chiral_restr0.0411689
ELECTRON MICROSCOPYf_plane_restr0.0041641

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