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- PDB-6wyl: Cryo-EM structure of GltPh L152C-G351C mutant in the intermediate... -

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Basic information

Database: PDB / ID: 6wyl
TitleCryo-EM structure of GltPh L152C-G351C mutant in the intermediate outward-facing state.
ComponentsGlutamate transporter homolog
KeywordsTRANSPORT PROTEIN / Glutamate transporter homolog Gltph
Function / homology
Function and homology information

amino acid:sodium symporter activity / L-aspartate transmembrane transport / L-aspartate import across plasma membrane / L-aspartate transmembrane transporter activity / chloride transmembrane transporter activity / chloride transmembrane transport / protein homotrimerization / integral component of membrane / identical protein binding / plasma membrane / metal ion binding
Sodium:dicarboxylate symporter, conserved site / Sodium:dicarboxylate symporter / Sodium:dicarboxylate symporter superfamily
Glutamate transporter homolog
Biological speciesPyrococcus horikoshii (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 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.
Validation Report
SummaryFull reportAbout validation report
DepositionMay 13, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 17, 2021Provider: 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

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Deposited unit
A: Glutamate transporter homolog
B: Glutamate transporter homolog
C: Glutamate transporter homolog
hetero molecules

Theoretical massNumber of molelcules
Total (without water)134,1546

TypeNameSymmetry operationNumber
identity operation1_5551


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

Mass: 44585.035 Da / Num. of mol.: 3 / Mutation: L152C, G351C, C321S
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 / Aspartic acid

Type: L-peptide linking / Mass: 133.103 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C4H7NO4
Has ligand of interestN

Experimental details


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

Sample preparation

ComponentName: Glutamate transporter homolog, GltPh, in nanodisc / 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
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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 FIELDBright-field microscopy
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)


SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
2EPUimage acquisition
4CTFFIND4.1CTF correction
10RELION3.07initial Euler assignment
11RELION3.07final Euler assignment
13RELION3.073D reconstruction
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 220938 / Symmetry type: POINT
RefinementHighest resolution: 3.9 Å
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0128991
ELECTRON MICROSCOPYf_angle_d0.87912234
ELECTRON MICROSCOPYf_dihedral_angle_d6.2891263
ELECTRON MICROSCOPYf_chiral_restr0.051560
ELECTRON MICROSCOPYf_plane_restr0.0061488

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