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- PDB-6wyj: Cryo-EM structure of the GltPh L152C-G321C mutant in the intermed... -

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Basic information

Entry
Database: PDB / ID: 6wyj
TitleCryo-EM structure of the GltPh L152C-G321C mutant in the intermediate state
ComponentsGlutamate transporter homolog
KeywordsTRANSPORT PROTEIN / Glutamate transporter homolog Gltph
Function / homology
Function and homology information


L-aspartate transmembrane transport / amino acid:sodium symporter activity / L-aspartate transmembrane transporter activity / L-aspartate import across plasma membrane / chloride transmembrane transporter activity / protein homotrimerization / chloride transmembrane transport / metal ion binding / identical protein binding / plasma membrane
Similarity search - Function
Proton glutamate symport protein / Sodium:dicarboxylate symporter / 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
ASPARTIC ACID / Glutamate transporter homolog
Similarity search - Component
Biological speciesPyrococcus horikoshii (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsFont, J. / Chen, I. / Sobti, M. / Stewart, A.G. / Ryan, R.M.
Funding support Australia, 1items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)APP1164494 Australia
CitationJournal: Nature / Year: 2021
Title: Glutamate transporters have a chloride channel with two hydrophobic gates.
Authors: Ichia Chen / Shashank Pant / Qianyi Wu / Rosemary J Cater / Meghna Sobti / Robert J Vandenberg / Alastair G Stewart / Emad Tajkhorshid / Josep Font / Renae M Ryan /
Abstract: Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, and its precise control is vital to maintain normal brain function and to prevent excitotoxicity. The removal ...Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, and its precise control is vital to maintain normal brain function and to prevent excitotoxicity. The removal of extracellular glutamate is achieved by plasma-membrane-bound transporters, which couple glutamate transport to sodium, potassium and pH gradients using an elevator mechanism. Glutamate transporters also conduct chloride ions by means of a channel-like process that is thermodynamically uncoupled from transport. However, the molecular mechanisms that enable these dual-function transporters to carry out two seemingly contradictory roles are unknown. Here we report the cryo-electron microscopy structure of a glutamate transporter homologue in an open-channel state, which reveals an aqueous cavity that is formed during the glutamate transport cycle. The functional properties of this cavity, combined with molecular dynamics simulations, reveal it to be an aqueous-accessible chloride permeation pathway that is gated by two hydrophobic regions and is conserved across mammalian and archaeal glutamate transporters. Our findings provide insight into the mechanism by which glutamate transporters support their dual function, and add information that will assist in mapping the complete transport cycle shared by the solute carrier 1A transporter family.
History
DepositionMay 13, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 17, 2021Provider: repository / Type: Initial release
Revision 1.0Feb 17, 2021Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 17, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 17, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Feb 17, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 17, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Feb 17, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 17, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Feb 17, 2021Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 17, 2021Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Mar 3, 2021Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Mar 24, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Mar 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 1.4May 14, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details / Item: _em_admin.last_update / _em_software.name
Revision 1.1May 14, 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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Assembly

Deposited unit
A: Glutamate transporter homolog
hetero molecules


Theoretical massNumber of molelcules
Total (without water)44,7182
Polymers44,5851
Non-polymers1331
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Glutamate transporter homolog / Glt(Ph) / Sodium-aspartate symporter Glt(Ph) / Sodium-dependent aspartate transporter


Mass: 44585.035 Da / Num. of mol.: 1 / Mutation: C321S, L152C, G351C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3) (archaea)
Strain: ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3
Gene: PH1295 / Production host: Escherichia coli (E. coli) / References: UniProt: O59010
#2: Chemical ChemComp-ASP / ASPARTIC ACID


Type: L-peptide linking / Mass: 133.103 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H7NO4
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: Monomer of Glutamate transporter homolog, GltPh, in intermediate configuration
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Pyrococcus horikoshii OT3 (archaea)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4.1CTF correction
8PHENIXmodel refinement
10RELION3.07initial Euler assignment
11RELION3.07final Euler assignment
13RELION3.073D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 105135 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.012997
ELECTRON MICROSCOPYf_angle_d0.6014078
ELECTRON MICROSCOPYf_dihedral_angle_d5.185421
ELECTRON MICROSCOPYf_chiral_restr0.048520
ELECTRON MICROSCOPYf_plane_restr0.003496

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