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- EMDB-12314: Structure of glutamate transporter homologue in complex with Sybody -

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Basic information

Entry
Database: EMDB / ID: EMD-12314
TitleStructure of glutamate transporter homologue in complex with Sybody
Map data
Sample
  • Complex: GltTk complex with sybody
    • Complex: Thermococcus kodakarensis KOD1
      • Protein or peptide: Proton/glutamate symporter, SDF family
    • Complex: synthetic construct
      • Protein or peptide: Sybody 1
  • 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 KOD1 (archaea) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsArkhipova V / Slotboom DJ / Guskov A
CitationJournal: Commun Biol / Year: 2021
Title: Kinetic mechanism of Na-coupled aspartate transport catalyzed by Glt.
Authors: Gianluca Trinco / Valentina Arkhipova / Alisa A Garaeva / Cedric A J Hutter / Markus A Seeger / Albert Guskov / Dirk J Slotboom /
Abstract: It is well-established that the secondary active transporters Glt and Glt catalyze coupled uptake of aspartate and three sodium ions, but insight in the kinetic mechanism of transport is fragmentary. ...It is well-established that the secondary active transporters Glt and Glt catalyze coupled uptake of aspartate and three sodium ions, but insight in the kinetic mechanism of transport is fragmentary. Here, we systematically measured aspartate uptake rates in proteoliposomes containing purified Glt, and derived the rate equation for a mechanism in which two sodium ions bind before and another after aspartate. Re-analysis of existing data on Glt using this equation allowed for determination of the turnover number (0.14 s), without the need for error-prone protein quantification. To overcome the complication that purified transporters may adopt right-side-out or inside-out membrane orientations upon reconstitution, thereby confounding the kinetic analysis, we employed a rapid method using synthetic nanobodies to inactivate one population. Oppositely oriented Glt proteins showed the same transport kinetics, consistent with the use of an identical gating element on both sides of the membrane. Our work underlines the value of bona fide transport experiments to reveal mechanistic features of Na-aspartate symport that cannot be observed in detergent solution. Combined with previous pre-equilibrium binding studies, a full kinetic mechanism of structurally characterized aspartate transporters of the SLC1A family is now emerging.
History
DepositionFeb 9, 2021-
Header (metadata) releaseSep 15, 2021-
Map releaseSep 15, 2021-
UpdateSep 15, 2021-
Current statusSep 15, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.131
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.131
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ngh
  • Surface level: 0.131
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7ngh
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12314.png

Downloads & links

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Map

FileDownload / File: emd_12314.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.012 Å
Density
Contour LevelBy AUTHOR: 0.131 / Movie #1: 0.131
Minimum - Maximum-0.17301509 - 0.4658582
Average (Standard dev.)0.00017323456 (±0.020368969)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 259.072 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0121.0121.012
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z259.072259.072259.072
α/β/γ90.00090.00090.000
start NX/NY/NZ1331310
NX/NY/NZ223226424
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1730.4660.000

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Supplemental data

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Sample components

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Entire : GltTk complex with sybody

EntireName: GltTk complex with sybody
Components
  • Complex: GltTk complex with sybody
    • Complex: Thermococcus kodakarensis KOD1
      • Protein or peptide: Proton/glutamate symporter, SDF family
    • Complex: synthetic construct
      • Protein or peptide: Sybody 1
  • Ligand: ASPARTIC ACID

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Supramolecule #1: GltTk complex with sybody

SupramoleculeName: GltTk complex with sybody / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: Thermococcus kodakarensis KOD1

SupramoleculeName: Thermococcus kodakarensis KOD1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Thermococcus kodakarensis KOD1 (archaea)

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Supramolecule #3: synthetic construct

SupramoleculeName: synthetic construct / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: Proton/glutamate symporter, SDF family

MacromoleculeName: Proton/glutamate symporter, SDF family / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Thermococcus kodakarensis KOD1 (archaea)
Molecular weightTheoretical: 46.40907 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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 IVGGVMQYAP 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 H

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Macromolecule #2: Sybody 1

MacromoleculeName: Sybody 1 / type: protein_or_peptide / ID: 2 / Details: Hexa His tag at C-terminus / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.862396 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSSSQVQLVE SGGGLVQAGG SLRLSCAASG FPVDSQFMHW YRQAPGKERE WVAAIESYGD ETYYADSVKG RFTISRDNAK NTVYLQMNS LKPEDTAVYY CRVLVGWGYY GQGTQVTVSA GRAGEQKLIS EEDLNSAVDH HHHHH

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Macromolecule #3: ASPARTIC ACID

MacromoleculeName: ASPARTIC ACID / type: ligand / ID: 3 / 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

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 8
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: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 53.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

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

6xwq

Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 53983

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