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- EMDB-21968: Cryo-EM structure of GltPh L152C-G351C mutant in the intermediate... -

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Basic information

Entry
Database: EMDB / ID: EMD-21968
TitleCryo-EM structure of GltPh L152C-G351C mutant in the intermediate outward-facing state.
Map data
SampleGlutamate transporter homolog, GltPh, in nanodisc:
Glutamate transporter homolog / ligand
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 OT3 (archaea) / Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3) (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsFont J / Chen I / Sobti M / Stewart AG / Ryan RM
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.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionMay 13, 2020-
Header (metadata) releaseFeb 17, 2021-
Map releaseFeb 17, 2021-
UpdateMar 24, 2021-
Current statusMar 24, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6wyl
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_21968.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.99 Å/pix.
x 256 pix.
= 252.416 Å
0.99 Å/pix.
x 256 pix.
= 252.416 Å
0.99 Å/pix.
x 256 pix.
= 252.416 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.986 Å
Density
Contour LevelBy AUTHOR: 0.027 / Movie #1: 0.03
Minimum - Maximum-0.10572847 - 0.19053744
Average (Standard dev.)-0.00016359205 (±0.0047030845)
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-0.1060.191-0.000

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Supplemental data

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Sample components

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Entire Glutamate transporter homolog, GltPh, in nanodisc

EntireName: Glutamate transporter homolog, GltPh, in nanodisc / Number of components: 3

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Component #1: protein, Glutamate transporter homolog, GltPh, in nanodisc

ProteinName: Glutamate transporter homolog, GltPh, in nanodisc / Recombinant expression: No
SourceSpecies: Pyrococcus horikoshii OT3 (archaea)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: protein, Glutamate transporter homolog

ProteinName: Glutamate transporter homolog / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 44.585035 kDa
SourceSpecies: 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
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: ligand, ASPARTIC ACID

LigandName: ASPARTIC ACID / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 0.133103 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
ImagingMicroscope: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 40 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: FEI FALCON III (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C3 (3 fold cyclic) / Number of projections: 220938
3D reconstructionSoftware: RELION / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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