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- EMDB-15393: Cryo-EM structure of a substrate-bound glutamate transporter homo... -

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Basic information

Entry
Database: EMDB / ID: EMD-15393
TitleCryo-EM structure of a substrate-bound glutamate transporter homologue GltTk encapsulated within a nanodisc
Map data
Sample
  • Complex: Glutamate transporter homologue GltTK in a lipid nanodisc environment bound with L-aspartate ligands
    • Protein or peptide: Proton/glutamate symporter, SDF family
  • Ligand: ASPARTIC ACID
Function / homologycarboxylic acid transport / Sodium:dicarboxylate symporter / Sodium:dicarboxylate symporter, conserved site / Sodium:dicarboxylate symporter superfamily / Sodium:dicarboxylate symporter family / Sodium:dicarboxylate symporter family signature 1. / symporter activity / membrane / Proton/glutamate symporter, SDF family
Function and homology information
Biological speciesThermococcus kodakarensis (archaea)
Methodsingle particle reconstruction / Resolution: 3.16 Å
AuthorsWhittaker JJ / Guskov A
Funding support Netherlands, 1 items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO) Netherlands
CitationJournal: Nat Commun / Year: 2020
Title: Structural ensemble of a glutamate transporter homologue in lipid nanodisc environment.
Authors: Valentina Arkhipova / Albert Guskov / Dirk J Slotboom /
Abstract: Glutamate transporters are cation-coupled secondary active membrane transporters that clear the neurotransmitter L-glutamate from the synaptic cleft. These transporters are homotrimers, with each ...Glutamate transporters are cation-coupled secondary active membrane transporters that clear the neurotransmitter L-glutamate from the synaptic cleft. These transporters are homotrimers, with each protomer functioning independently by an elevator-type mechanism, in which a mobile transport domain alternates between inward- and outward-oriented states. Using single-particle cryo-EM we have determined five structures of the glutamate transporter homologue Glt, a Na- L-aspartate symporter, embedded in lipid nanodiscs. Dependent on the substrate concentrations used, the protomers of the trimer adopt a variety of asymmetrical conformations, consistent with the independent movement. Six of the 15 resolved protomers are in a hitherto elusive state of the transport cycle in which the inward-facing transporters are loaded with Na ions. These structures explain how substrate-leakage is prevented - a strict requirement for coupled transport. The belt protein of the lipid nanodiscs bends around the inward oriented protomers, suggesting that membrane deformations occur during transport.
History
DepositionJul 16, 2022-
Header (metadata) releaseApr 5, 2023-
Map releaseApr 5, 2023-
UpdateApr 12, 2023-
Current statusApr 12, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15393.png

Downloads & links

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Map

FileDownload / File: emd_15393.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 256 pix.
= 214.016 Å		0.84 Å/pix.
x 256 pix.
= 214.016 Å		0.84 Å/pix.
x 256 pix.
= 214.016 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.836 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.112
Minimum - Maximum-1.4631937 - 2.6295235
Average (Standard dev.)0.012032687 (±0.063896306)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 214.016 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_15393_half_map_1.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15393_half_map_2.map
Projections & Slices
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Sample components

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Entire : Glutamate transporter homologue GltTK in a lipid nanodisc environ...

EntireName: Glutamate transporter homologue GltTK in a lipid nanodisc environment bound with L-aspartate ligands
Components
  • Complex: Glutamate transporter homologue GltTK in a lipid nanodisc environment bound with L-aspartate ligands
    • Protein or peptide: Proton/glutamate symporter, SDF family
  • Ligand: ASPARTIC ACID

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Supramolecule #1: Glutamate transporter homologue GltTK in a lipid nanodisc environ...

SupramoleculeName: Glutamate transporter homologue GltTK in a lipid nanodisc environment bound with L-aspartate ligands
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Thermococcus kodakarensis (archaea) / Strain: BL21(DE3)
Molecular weightTheoretical: 1.39 MDa

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Macromolecule #1: Proton/glutamate symporter, SDF family

MacromoleculeName: Proton/glutamate symporter, SDF family / type: protein_or_peptide / ID: 1 / Details: Mutant P208R / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Thermococcus kodakarensis (archaea) / Strain: ATCC BAA-918 / JCM 12380 / KOD1 / Cell: E.coli
Molecular weightTheoretical: 46.745441 KDa
Recombinant expressionOrganism: Thermococcus kodakarensis (archaea)
SequenceString: MGKSLLRRYL DYPVLWKILW GLVLGAVFGL IAGHFGYAGA VKTYIKPFGD LFVRLLKMLV MPIVLASLVV GAASISPARL GRVGVKIVV YYLATSAMAV FFGLIVGRLF NVGANVNLGS GTGKAIEAQP PSLVQTLLNI VPTNPFASLA KGEVLPVIFF A IILGIAIT ...String:
MGKSLLRRYL DYPVLWKILW GLVLGAVFGL IAGHFGYAGA VKTYIKPFGD LFVRLLKMLV MPIVLASLVV GAASISPARL GRVGVKIVV YYLATSAMAV FFGLIVGRLF NVGANVNLGS GTGKAIEAQP PSLVQTLLNI VPTNPFASLA KGEVLPVIFF A IILGIAIT YLMNRNEERV RKSAETLLRV FDGLAEAMYL IVGGVMQYAR IGVFALIAYV MAEQGVRVVG PLAKVVGAVY TG LFLQIVI TYFILLKVFG IDPIKFIRKA KDAMITAFVT RSSSGTLPVT MRVAEEEMGV DKGIFSFTLP LGATINMDGT ALY QGVTVL FVANAIGHPL TLGQQLVVVL TAVLASIGTA GVPGAGAIML AMVLQSVGLD LTPGSPVALA YAMILGIDAI LDMG RTMVN VTGDLAGTVI VAKTEKELDE SKWISHHHHH HHH

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Macromolecule #2: ASPARTIC ACID

MacromoleculeName: ASPARTIC ACID / type: ligand / ID: 2 / Number of copies: 3 / 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

Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration6.5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMC4H11NO3tris(hydroxymethyl)aminomethane
300.0 mMNaClSodium chloridepotassium chloride
0.01 mMC22H44O11Decyl maltoside
GridModel: Quantifoil R2/4 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 100.0 nm

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Electron microscopy

MicroscopeTFS TALOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: KODAK 4489 FILM / Digitization - Scanner: TEMSCAN / Average electron dose: 50.0 e/Å2

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5e9s

Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.16 Å / Resolution method: FSC 0.33 CUT-OFF / Number images used: 3000
FSC plot (resolution estimation)

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