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- PDB-6x01: Crystal structure of the GltPh V216C-A391C mutant cross-linked in... -

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Basic information

Entry
Database: PDB / ID: 6x01
TitleCrystal structure of the GltPh V216C-A391C mutant cross-linked in outward-facing state
ComponentsGlutamate transporter homolog
KeywordsTRANSPORT PROTEIN / Glutamate transporter homolog
Function / homology
Function and homology information


amino acid:sodium symporter activity / L-aspartate transmembrane transport / L-aspartate transmembrane transporter activity / L-aspartate import across plasma membrane / chloride transmembrane transporter activity / protein homotrimerization / chloride transmembrane transport / identical protein binding / metal ion binding / plasma membrane
Similarity search - Function
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
ASPARTIC ACID / Glutamate transporter homolog
Similarity search - Component
Biological speciesPyrococcus horikoshii (archaea)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.65 Å
AuthorsChen, I. / Font, J. / Ryan, R.
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 15, 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
Revision 1.3Oct 18, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)134,29212
Polymers133,7553
Non-polymers5379
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6970 Å2
ΔGint-109 kcal/mol
Surface area45290 Å2
MethodPISA
Unit cell
Length a, b, c (Å)113.985, 113.985, 321.705
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number169
Space group name H-MP61
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11chain A
21chain B
31chain C

NCS domain segments:

Component-ID: 1 / Ens-ID: 1 / Beg auth comp-ID: VAL / Beg label comp-ID: VAL / End auth comp-ID: GLU / End label comp-ID: GLU / Auth seq-ID: 12 - 416 / Label seq-ID: 12 - 416

Dom-IDSelection detailsAuth asym-IDLabel asym-ID
1chain AAA
2chain BBB
3chain CCC

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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.: 3 / Mutation: V216C, A391C, 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


Type: L-peptide linking / Mass: 133.103 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C4H7NO4
#3: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Na
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 4.51 Å3/Da / Density % sol: 72.73 %
Crystal growTemperature: 277.15 K / Method: vapor diffusion, hanging drop
Details: 15/23% PEG 1000, 100mM Lithium sulfate, 50mM citric acid, 50mM Disodium phosphate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 0.9537 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: May 6, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9537 Å / Relative weight: 1
ReflectionResolution: 3.65→48.79 Å / Num. obs: 26118 / % possible obs: 100 % / Redundancy: 21.6 % / CC1/2: 0.999 / Rmerge(I) obs: 0.128 / Net I/σ(I): 14
Reflection shellResolution: 3.65→3.9 Å / Rmerge(I) obs: 3.559 / Mean I/σ(I) obs: 1.2 / Num. unique obs: 4731 / CC1/2: 0.851

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Processing

Software
NameVersionClassification
PHENIX1.18.2_3874refinement
Aimlessdata scaling
PDB_EXTRACT3.25data extraction
XDSdata reduction
Auto-Rickshawphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2NWX

2nwx


Resolution: 3.65→44.84 Å / SU ML: 0.59 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 45.85 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.3086 1227 4.7 %
Rwork0.2723 24891 -
obs0.2733 26118 99.69 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 280.65 Å2 / Biso mean: 176.7668 Å2 / Biso min: 76.74 Å2
Refinement stepCycle: final / Resolution: 3.65→44.84 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8829 0 33 0 8862
Biso mean--155.05 --
Num. residues----1200
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A3609X-RAY DIFFRACTION0.571TORSIONAL
12B3609X-RAY DIFFRACTION0.571TORSIONAL
13C3609X-RAY DIFFRACTION0.571TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 9

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
3.65-3.80.43261580.36052741289999
3.8-3.970.33061250.304427382863100
3.97-4.180.31441430.281927552898100
4.18-4.440.2941460.243127562902100
4.44-4.780.27271180.226927962914100
4.78-5.260.29191330.269827502883100
5.26-6.020.35961490.314327742923100
6.02-7.580.27771290.286327942923100
7.58-44.840.30291260.260527872913100

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