+Open data
-Basic information
Entry | Database: PDB / ID: 7t9g | ||||||||||||||||||
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Title | Structure of VcINDY-Na+ | ||||||||||||||||||
Components | DASS family sodium-coupled anion symporter | ||||||||||||||||||
Keywords | MEMBRANE PROTEIN / TRANSPORTER | ||||||||||||||||||
Function / homology | citrate transmembrane transporter activity / Citrate transporter-like domain / Citrate transporter / Sodium/sulphate symporter, conserved site / Sodium:sulfate symporter family signature. / Solute carrier family 13 / succinate transmembrane transporter activity / membrane / Transporter, NadC family Function and homology information | ||||||||||||||||||
Biological species | Vibrio cholerae (bacteria) | ||||||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.83 Å | ||||||||||||||||||
Authors | Sauer, D.B. / Marden, J.J. / Song, J.M. / Wang, D.N. | ||||||||||||||||||
Funding support | United States, 5items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7t9g.cif.gz | 287.2 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7t9g.ent.gz | 234.5 KB | Display | PDB format |
PDBx/mmJSON format | 7t9g.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7t9g_validation.pdf.gz | 1.5 MB | Display | wwPDB validaton report |
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Full document | 7t9g_full_validation.pdf.gz | 1.5 MB | Display | |
Data in XML | 7t9g_validation.xml.gz | 37.9 KB | Display | |
Data in CIF | 7t9g_validation.cif.gz | 55.7 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/t9/7t9g ftp://data.pdbj.org/pub/pdb/validation_reports/t9/7t9g | HTTPS FTP |
-Related structure data
Related structure data | 25757MC 7t9fC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
EM raw data | EMPIAR-10969 (Title: Particle stacks for VcINDY in 300mM NaCl / Data size: 35.4 Data #1: Particle stack of VcINDY in 300 mM NaCl [picked particles - multiframe - processed]) |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | 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 #2: Chemical | ChemComp-NA / #3: Water | ChemComp-HOH / | Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: VcINDY in the Ci-Na+ state / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: Vibrio cholerae (bacteria) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.5 |
Specimen | Conc.: 1.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: GOLD / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm |
Image recording | Average 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: 4253 |
-Processing
Software | Name: PHENIX / Version: 1.19.2_4158: / Classification: refinement | |||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1520631 | |||||||||||||||||||||||||||
Symmetry | Point symmetry: C2 (2 fold cyclic) | |||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.83 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 144865 / Symmetry type: POINT | |||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL | |||||||||||||||||||||||||||
Atomic model building | PDB-ID: 5UL9 Accession code: 5UL9 / Source name: PDB / Type: experimental model | |||||||||||||||||||||||||||
Refine LS restraints |
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