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- PDB-6wu4: Structure of the LaINDY-alpha-ketoglutarate complex -

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Basic information

Entry
Database: PDB / ID: 6wu4
TitleStructure of the LaINDY-alpha-ketoglutarate complex
ComponentsDASS family sodium-coupled anion symporter
KeywordsMEMBRANE PROTEIN / Transporter / Structural Genomics / PSI-Biology / New York Consortium on Membrane Protein Structure / NYCOMPS
Function / homologyCitrate carrier CitT-related / Sodium:sulfate symporter transmembrane region / Solute carrier family 13 / transmembrane transporter activity / membrane => GO:0016020 / membrane / DASS family sodium-coupled anion symporter / 2-oxoglutarate-malate translocator
Function and homology information
Biological speciesLactobacillus acidophilus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.71 Å
AuthorsSauer, D.B. / Marden, J.J. / Cocco, N. / Song, J.M. / Wang, D.N. / New York Consortium on Membrane Protein Structure (NYCOMPS)
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U54GM095315 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS108151 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM121994 United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)R01DK099023 United States
CitationJournal: Elife / Year: 2020
Title: Structural basis for the reaction cycle of DASS dicarboxylate transporters.
Authors: David B Sauer / Noah Trebesch / Jennifer J Marden / Nicolette Cocco / Jinmei Song / Akiko Koide / Shohei Koide / Emad Tajkhorshid / Da-Neng Wang /
Abstract: Citrate, α-ketoglutarate and succinate are TCA cycle intermediates that also play essential roles in metabolic signaling and cellular regulation. These di- and tricarboxylates are imported into the ...Citrate, α-ketoglutarate and succinate are TCA cycle intermediates that also play essential roles in metabolic signaling and cellular regulation. These di- and tricarboxylates are imported into the cell by the divalent anion sodium symporter (DASS) family of plasma membrane transporters, which contains both cotransporters and exchangers. While DASS proteins transport substrates via an elevator mechanism, to date structures are only available for a single DASS cotransporter protein in a substrate-bound, inward-facing state. We report multiple cryo-EM and X-ray structures in four different states, including three hitherto unseen states, along with molecular dynamics simulations, of both a cotransporter and an exchanger. Comparison of these outward- and inward-facing structures reveal how the transport domain translates and rotates within the framework of the scaffold domain through the transport cycle. Additionally, we propose that DASS transporters ensure substrate coupling by a charge-compensation mechanism, and by structural changes upon substrate release.
History
DepositionMay 4, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 16, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: DASS family sodium-coupled anion symporter
B: DASS family sodium-coupled anion symporter


Theoretical massNumber of molelcules
Total (without water)113,1282
Polymers113,1282
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area5640 Å2
ΔGint-55 kcal/mol
Surface area37660 Å2

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Components

#1: Protein DASS family sodium-coupled anion symporter


Mass: 56563.758 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactobacillus acidophilus (bacteria) / Gene: D7H66_01865 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A3A9Y7Q2, UniProt: Q5FKK5*PLUS

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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

ComponentName: LaINDY-alpha-ketoglutarate complex / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Lactobacillus acidophilus (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 FIELD / Nominal magnification: 36000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm
Image recordingAverage exposure time: 2.8 sec. / Electron dose: 46.13 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC2.12particle selection
2Leginonimage acquisition
4cryoSPARC2.12CTF correction
7PHENIXmodel fitting
9PHENIXmodel refinement
10cryoSPARC2.12initial Euler assignment
11cryoSPARC2.12final Euler assignment
12cryoSPARC2.12classification
13cryoSPARC2.123D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.71 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 64216 / Symmetry type: POINT

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