[English] 日本語
Yorodumi
- PDB-7npw: Cryo-EM structure of Human excitatory amino acid transporters-1 (... -

+
Open data


ID or keywords:

Loading...

-
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) / Astrocytic Glutamate-Glutamine Uptake And Metabolism / cranial nerve development / gamma-aminobutyric acid biosynthetic process / neurotransmitter uptake / cell morphogenesis involved in neuron differentiation / membrane protein complex / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity / L-glutamate import ...Defective SLC1A3 causes episodic ataxia 6 (EA6) / Astrocytic Glutamate-Glutamine Uptake And Metabolism / cranial nerve development / gamma-aminobutyric acid biosynthetic process / neurotransmitter uptake / cell morphogenesis involved in neuron differentiation / membrane protein complex / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity / L-glutamate import / : / L-glutamate transmembrane transporter activity / L-glutamate transmembrane transport / D-aspartate import across plasma membrane / L-aspartate import across plasma membrane / Glutamate Neurotransmitter Release Cycle / neutral L-amino acid transmembrane transporter activity / auditory behavior / L-glutamate import across plasma membrane / transepithelial transport / intracellular sodium ion homeostasis / cellular response to cocaine / glutamate binding / neuromuscular process controlling balance / neurotransmitter transport / response to light stimulus / positive regulation of synaptic transmission / transport across blood-brain barrier / 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 / neuron projection / response to xenobiotic stimulus / response to antibiotic / neuronal cell body / synapse / perinuclear region of cytoplasm / cell surface / metal ion 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
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

-
Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
  • Download
  • Superimposition on EM map
  • EMDB-12524
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
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

-
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

-
Experimental details

-
Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

-
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

-
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

-
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

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more