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- PDB-7ngh: Structure of glutamate transporter homologue in complex with Sybody -

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Basic information

Entry
Database: PDB / ID: 7ngh
TitleStructure of glutamate transporter homologue in complex with Sybody
Components
  • Proton/glutamate symporter, SDF family
  • Sybody 1
KeywordsTRANSPORT PROTEIN / glutamate transporter homologue / GltTk / amino acid transport / membrane protein
Function / homology
Function and homology information


carboxylic acid transport / symporter activity / 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
ASPARTIC ACID / Proton/glutamate symporter, SDF family
Similarity search - Component
Biological speciesThermococcus kodakarensis KOD1 (archaea)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsArkhipova, V. / Slotboom, D.J. / 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, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 15, 2021Provider: repository / Type: Initial release

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Assembly

Deposited unit
A: Proton/glutamate symporter, SDF family
B: Proton/glutamate symporter, SDF family
C: Proton/glutamate symporter, SDF family
D: Sybody 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)155,4897
Polymers155,0904
Non-polymers3993
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area8710 Å2
ΔGint-73 kcal/mol
Surface area52380 Å2

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Components

#1: Protein Proton/glutamate symporter, SDF family


Mass: 46409.070 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus kodakarensis KOD1 (archaea)
Gene: TK0986 / Production host: Escherichia coli (E. coli) / References: UniProt: Q5JID0
#2: Protein Sybody 1


Mass: 15862.396 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Hexa His tag at C-terminus / Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#3: Chemical ChemComp-ASP / ASPARTIC ACID / Aspartic acid


Type: L-peptide linking / Mass: 133.103 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C4H7NO4
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1GltTk complex with sybodyCOMPLEX#1-#20MULTIPLE SOURCES
2Thermococcus kodakarensis KOD1COMPLEX#11NATURAL
3synthetic constructCOMPLEX#21SYNTHETIC
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Thermococcus kodakarensis KOD1 (archaea)69014
33synthetic construct (others)32630
Buffer solutionpH: 8
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 53 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 53983 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00510508
ELECTRON MICROSCOPYf_angle_d0.59914295
ELECTRON MICROSCOPYf_dihedral_angle_d7.7366181
ELECTRON MICROSCOPYf_chiral_restr0.041759
ELECTRON MICROSCOPYf_plane_restr0.0041760

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