[English] 日本語
![](img/lk-miru.gif)
- EMDB-12314: Structure of glutamate transporter homologue in complex with Sybody -
+
Open data
-
Basic information
Entry | Database: EMDB / ID: EMD-12314 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | Structure of glutamate transporter homologue in complex with Sybody | |||||||||
![]() | ||||||||||
![]() |
| |||||||||
Function / homology | carboxylic 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![]() | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.5 Å | |||||||||
![]() | Arkhipova V / Slotboom DJ / Guskov A | |||||||||
![]() | ![]() 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 |
|
-
Structure visualization
Movie |
![]() |
---|---|
Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
-
Downloads & links
-EMDB archive
Map data | ![]() | 32.1 MB | ![]() | |
---|---|---|---|---|
Header (meta data) | ![]() ![]() | 11.5 KB 11.5 KB | Display Display | ![]() |
Images | ![]() | 51.6 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7nghMC M: atomic model generated by this map C: citing same article ( |
---|---|
Similar structure data |
-
Links
EMDB pages | ![]() ![]() |
---|---|
Related items in Molecule of the Month |
-
Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Voxel size | X=Y=Z: 1.012 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
|
-Supplemental data
-
Sample components
-Entire : GltTk complex with sybody
Entire | Name: GltTk complex with sybody |
---|---|
Components |
|
-Supramolecule #1: GltTk complex with sybody
Supramolecule | Name: GltTk complex with sybody / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 |
---|
-Supramolecule #2: Thermococcus kodakarensis KOD1
Supramolecule | Name: Thermococcus kodakarensis KOD1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
---|---|
Source (natural) | Organism: ![]() ![]() |
-Supramolecule #3: synthetic construct
Supramolecule | Name: synthetic construct / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 |
---|---|
Source (natural) | Organism: synthetic construct (others) |
-Macromolecule #1: Proton/glutamate symporter, SDF family
Macromolecule | Name: Proton/glutamate symporter, SDF family / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO |
---|---|
Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 46.40907 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 |
-Macromolecule #2: Sybody 1
Macromolecule | Name: 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 weight | Theoretical: 15.862396 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: GSSSQVQLVE SGGGLVQAGG SLRLSCAASG FPVDSQFMHW YRQAPGKERE WVAAIESYGD ETYYADSVKG RFTISRDNAK NTVYLQMNS LKPEDTAVYY CRVLVGWGYY GQGTQVTVSA GRAGEQKLIS EEDLNSAVDH HHHHH |
-Macromolecule #3: ASPARTIC ACID
Macromolecule | Name: ASPARTIC ACID / type: ligand / ID: 3 / Number of copies: 3 / Formula: ASP |
---|---|
Molecular weight | Theoretical: 133.103 Da |
Chemical component information | ![]() ChemComp-ASP: |
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
![]() | single particle reconstruction |
Aggregation state | particle |
-
Sample preparation
Concentration | 1 mg/mL |
---|---|
Buffer | pH: 8 |
Vitrification | Cryogen name: ETHANE |
-
Electron microscopy
Microscope | FEI TALOS ARCTICA |
---|---|
Image recording | Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 53.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Experimental equipment | ![]() Model: Talos Arctica / Image courtesy: FEI Company |
-
Image processing
Startup model | Type of model: PDB ENTRY PDB model - PDB ID: |
---|---|
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 53983 |
Initial angle assignment | Type: NOT APPLICABLE |
Final angle assignment | Type: NOT APPLICABLE |