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Yorodumi- PDB-7npw: Cryo-EM structure of Human excitatory amino acid transporters-1 (... -
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-Basic information
Entry | Database: PDB / ID: 7npw | ||||||
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Title | Cryo-EM structure of Human excitatory amino acid transporters-1 (EAAT1) in potassium buffer | ||||||
Components | Excitatory amino acid transporter 1 | ||||||
Keywords | MEMBRANE PROTEIN / Human Membrane Protein / Transporter / Glutamate transporter | ||||||
Function / homology | Function and homology information Defective SLC1A3 causes episodic ataxia 6 (EA6) / Astrocytic Glutamate-Glutamine Uptake And Metabolism / auditory behavior / 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) / Astrocytic Glutamate-Glutamine Uptake And Metabolism / auditory behavior / 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 / Glutamate Neurotransmitter Release Cycle / neutral L-amino acid transmembrane transporter activity / 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 / basal plasma membrane / chloride transmembrane transport / potassium ion transmembrane transport / sensory perception of sound / response to wounding / cytoplasmic vesicle / chemical synaptic transmission / neuron projection / response to xenobiotic stimulus / response to antibiotic / neuronal cell body / synapse / perinuclear region of cytoplasm / cell surface / membrane / metal ion binding / plasma membrane Similarity search - Function | ||||||
Biological species | Homo sapiens (human) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.99 Å | ||||||
Authors | Kumar, A. / Reyes, N. | ||||||
Funding support | France, 1items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7npw.cif.gz | 224.8 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7npw.ent.gz | 182 KB | Display | PDB format |
PDBx/mmJSON format | 7npw.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7npw_validation.pdf.gz | 840.2 KB | Display | wwPDB validaton report |
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Full document | 7npw_full_validation.pdf.gz | 857.6 KB | Display | |
Data in XML | 7npw_validation.xml.gz | 40.1 KB | Display | |
Data in CIF | 7npw_validation.cif.gz | 59.7 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/np/7npw ftp://data.pdbj.org/pub/pdb/validation_reports/np/7npw | HTTPS FTP |
-Related structure data
Related structure data | 12524MC 7awlC 7awmC 7awnC 7awpC 7awqC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | 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
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Human excitatory amino acid transporter 1 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT | ||||||||||||||||||||||||||||||
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Molecular weight | Experimental value: NO | ||||||||||||||||||||||||||||||
Source (natural) | Organism: Homo sapiens (human) | ||||||||||||||||||||||||||||||
Source (recombinant) | Organism: Homo sapiens (human) / Cell: HEK-293F / Plasmid: pcDNA3.1 | ||||||||||||||||||||||||||||||
Buffer solution | pH: 7.4 | ||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 | ||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: Grids were blotted for 4 Sec |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm |
Specimen holder | Cryogen: NITROGEN |
Image recording | Electron 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 scans | Movie frames/image: 40 / Used frames/image: 1-40 |
-Processing
Software | Name: PHENIX / Version: 1.18.2_3874: / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Details: Gctf / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 2298481 | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C3 (3 fold cyclic) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.99 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 34433 / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: 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 |
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