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- EMDB-21966: Cryo-EM structure of the GltPh L152C-G321C mutant in the intermed... -

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Basic information

Entry
Database: EMDB / ID: EMD-21966
TitleCryo-EM structure of the GltPh L152C-G321C mutant in the intermediate state.
Map dataCryo-EM structure of the GltPh L152C-G321C mutant in the intermediate state
Sample
  • Complex: Monomer of Glutamate transporter homolog, GltPh, in intermediate configuration
    • Protein or peptide: Glutamate transporter homolog
  • Ligand: ASPARTIC ACID
KeywordsGlutamate transporter homolog Gltph / TRANSPORT PROTEIN
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
Glutamate transporter homolog
Similarity search - Component
Biological speciesPyrococcus horikoshii OT3 (archaea) / Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3) (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsFont J / Chen I
Funding support Australia, 1 items
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, 2020-
Header (metadata) releaseFeb 17, 2021-
Map releaseFeb 17, 2021-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6wyj
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6wyj
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21966.png

Downloads & links

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Map

FileDownload / File: emd_21966.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of the GltPh L152C-G321C mutant in the intermediate state
Voxel sizeX=Y=Z: 0.986 Å
Density
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.07
Minimum - Maximum-0.37747845 - 0.54733783
Average (Standard dev.)0.000084560124 (±0.0072406186)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 252.416 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.9860.9860.986
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z252.416252.416252.416
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.3770.5470.000

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Supplemental data

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Sample components

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Entire : Monomer of Glutamate transporter homolog, GltPh, in intermediate ...

EntireName: Monomer of Glutamate transporter homolog, GltPh, in intermediate configuration
Components
  • Complex: Monomer of Glutamate transporter homolog, GltPh, in intermediate configuration
    • Protein or peptide: Glutamate transporter homolog
  • Ligand: ASPARTIC ACID

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Supramolecule #1: Monomer of Glutamate transporter homolog, GltPh, in intermediate ...

SupramoleculeName: Monomer of Glutamate transporter homolog, GltPh, in intermediate configuration
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Pyrococcus horikoshii OT3 (archaea)

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Macromolecule #1: Glutamate transporter homolog

MacromoleculeName: Glutamate transporter homolog / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: 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
Molecular weightTheoretical: 44.585035 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGLYRKYIEY PVLQKILIGL ILGAIVGLIL GHYGYADAVK TYVKPFGDLF VRLLKMLVMP IVFASLVVGA ASISPARLGR VGVKIVVYY LLTSAFAVTL GIIMARLFNP GAGIHLAVGG QQFQPKQAPP LVKILLDIVP TNPFGALANG QVCPTIFFAI I LGIAITYL ...String:
MGLYRKYIEY PVLQKILIGL ILGAIVGLIL GHYGYADAVK TYVKPFGDLF VRLLKMLVMP IVFASLVVGA ASISPARLGR VGVKIVVYY LLTSAFAVTL GIIMARLFNP GAGIHLAVGG QQFQPKQAPP LVKILLDIVP TNPFGALANG QVCPTIFFAI I LGIAITYL MNSENEKVRK SAETLLDAIN GLAEAMYKIV NGVMQYAPIG VFALIAYVMA EQGVKVVGEL AKVTAAVYVG LT LQILLVY FVLLKIYGID PISFIKKAKD AMLTAFVTRS SSGTLPVTMR VAKEMGISEG IYSFTLPLGA TINMDGTALY QGV STFFIA NALGSHLTVG QQLTIVLTAV LASICTAGVP GAGAIMLAMV LESVGLPLTD PNVAAAYAMI LGIDAILDMG RTMV NVTGD LTGTAIVAKT EGTLVPR

UniProtKB: Glutamate transporter homolog

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Macromolecule #2: ASPARTIC ACID

MacromoleculeName: ASPARTIC ACID / type: ligand / ID: 2 / Number of copies: 1 / Formula: ASP
Molecular weightTheoretical: 133.103 Da
Chemical component information

ChemComp-ASP:
ASPARTIC ACID / Aspartic acid

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: RANDOM CONICAL TILT
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.07)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.07)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.07) / Number images used: 105135

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