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- PDB-6wu1: Structure of apo LaINDY -

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Basic information

Entry
Database: PDB / ID: 6wu1
TitleStructure of apo LaINDY
ComponentsDASS family sodium-coupled anion symporter
KeywordsMEMBRANE PROTEIN / Transporter / Structural Genomics / PSI-Biology / New York Consortium on Membrane Protein Structure / NYCOMPS
Function / homologySolute carrier family 13 / Citrate carrier CitT-related / transmembrane transporter activity / integral component of membrane / DASS family sodium-coupled anion symporter
Function and homology information
Biological speciesLactobacillus acidophilus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.09 Å
AuthorsSauer, D.B. / Marden, J.J. / Cocco, N.C. / 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) 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.
Validation Report
SummaryFull reportAbout validation report
History
DepositionMay 4, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 16, 2020Provider: repository / Type: Initial release

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)115,81326
Polymers113,1282
Non-polymers2,68524
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area13240 Å2
ΔGint67 kcal/mol
Surface area36000 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
#2: Chemical
ChemComp-HEX / HEXANE / Hexane


Mass: 86.175 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C6H14
#3: Chemical
ChemComp-D10 / DECANE / Decane


Mass: 142.282 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H22
#4: Chemical
ChemComp-OCT / N-OCTANE / Octane


Mass: 114.229 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C8H18
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

ComponentName: LaINDY / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / 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: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm
Image recordingAverage exposure time: 10 sec. / Electron dose: 75.05 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3122

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Processing

EM software
IDNameVersionCategory
1cryoSPARC2.12particle selection
2Leginonimage acquisition
4cryoSPARC2.12CTF correction
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: 3.09 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 278663 / Symmetry type: POINT

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