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- PDB-7t9f: Structure of VcINDY-apo -

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Basic information

Entry
Database: PDB / ID: 7t9f
TitleStructure of VcINDY-apo
ComponentsDASS family sodium-coupled anion symporter
KeywordsMEMBRANE PROTEIN / TRANSPORTER
Function / homologyCitrate transporter-like domain / Citrate transporter / Sodium/sulphate symporter, conserved site / Sodium:sulfate symporter family signature. / succinate transmembrane transporter activity / Solute carrier family 13 / plasma membrane / Transporter, NadC family
Function and homology information
Biological speciesVibrio cholerae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.23 Å
AuthorsSauer, D.B. / Marden, J.J. / Song, J.M. / Wang, D.N.
Funding support United States, 5items
OrganizationGrant numberCountry
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
American Cancer Society129844-PF-17-135-01-TBE United States
Department of Defense (DOD, United States)W81XWH-16-1-0153 United States
CitationJournal: Nat Commun / Year: 2022
Title: Structural basis of ion - substrate coupling in the Na-dependent dicarboxylate transporter VcINDY.
Authors: David B Sauer / Jennifer J Marden / Joseph C Sudar / Jinmei Song / Christopher Mulligan / Da-Neng Wang /
Abstract: The Na-dependent dicarboxylate transporter from Vibrio cholerae (VcINDY) is a prototype for the divalent anion sodium symporter (DASS) family. While the utilization of an electrochemical Na gradient ...The Na-dependent dicarboxylate transporter from Vibrio cholerae (VcINDY) is a prototype for the divalent anion sodium symporter (DASS) family. While the utilization of an electrochemical Na gradient to power substrate transport is well established for VcINDY, the structural basis of this coupling between sodium and substrate binding is not currently understood. Here, using a combination of cryo-EM structure determination, succinate binding and site-directed cysteine alkylation assays, we demonstrate that the VcINDY protein couples sodium- and substrate-binding via a previously unseen cooperative mechanism by conformational selection. In the absence of sodium, substrate binding is abolished, with the succinate binding regions exhibiting increased flexibility, including HPb, TM10b and the substrate clamshell motifs. Upon sodium binding, these regions become structurally ordered and create a proper binding site for the substrate. Taken together, these results provide strong evidence that VcINDY's conformational selection mechanism is a result of the sodium-dependent formation of the substrate binding site.
History
DepositionDec 19, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 25, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 28, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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)96,3152
Polymers96,3152
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein DASS family sodium-coupled anion symporter / Transporter / NadC family


Mass: 48157.359 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae (bacteria) / Gene: VC0395_0108, GG844_00510 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0H3AG83

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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: VcINDY in the Ci-apo state / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Vibrio cholerae (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 1.3 mg/ml / Embedding 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 FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Image recordingAverage exposure time: 2.34 sec. / Electron dose: 65 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4514

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameCategory
1cryoSPARCparticle selection
4cryoSPARCCTF correction
7PHENIXmodel fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6100049
3D reconstructionResolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 134038 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 5UL9

5ul9


Accession code: 5UL9 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0046840
ELECTRON MICROSCOPYf_angle_d0.8189326
ELECTRON MICROSCOPYf_dihedral_angle_d4.873908
ELECTRON MICROSCOPYf_chiral_restr0.0921158
ELECTRON MICROSCOPYf_plane_restr0.0041128

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