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- EMDB-25757: Structure of VcINDY-Na+ -

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Basic information

Entry
Database: EMDB / ID: EMD-25757
TitleStructure of VcINDY-Na+
Map dataPhenix Autosharpened map
Sample
  • Organelle or cellular component: VcINDY in the Ci-Na+ state
    • Protein or peptide: DASS family sodium-coupled anion symporter
  • Ligand: SODIUM IONSodium
  • Ligand: water
KeywordsTRANSPORTER / MEMBRANE PROTEIN
Function / homologyCitrate 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 speciesVibrio cholerae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.83 Å
AuthorsSauer DB / Marden JJ
Funding support United States, 5 items
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, 2021-
Header (metadata) releaseMay 25, 2022-
Map releaseMay 25, 2022-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25757.png

Downloads & links

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Map

FileDownload / File: emd_25757.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPhenix Autosharpened map
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 5.12
Minimum - Maximum-10.959965 - 23.743835000000001
Average (Standard dev.)0.000000000005404 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 212.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened map

Fileemd_25757_additional_1.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Halfmap B

Fileemd_25757_half_map_1.map
AnnotationHalfmap B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Halfmap A

Fileemd_25757_half_map_2.map
AnnotationHalfmap A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : VcINDY in the Ci-Na+ state

EntireName: VcINDY in the Ci-Na+ state
Components
  • Organelle or cellular component: VcINDY in the Ci-Na+ state
    • Protein or peptide: DASS family sodium-coupled anion symporter
  • Ligand: SODIUM IONSodium
  • Ligand: water

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Supramolecule #1: VcINDY in the Ci-Na+ state

SupramoleculeName: VcINDY in the Ci-Na+ state / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Vibrio cholerae (bacteria)

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Macromolecule #1: DASS family sodium-coupled anion symporter

MacromoleculeName: DASS family sodium-coupled anion symporter / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 48.157359 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: REWFLHRNSL IVLADVALFL ALYHFLPFEH NVVLGISMLA FIAVLWLTEA LHVTVTAILV PVMAVFFGIF ETQAALNNFA NSIIFLFLG GFALAAAMHH QGLDKVIADK VLAMAQGKMS VAVFMLFGVT ALLSMWISNT ATAAMMLPLV LGVLSKVDAD K QRSTYVFV ...String:
REWFLHRNSL IVLADVALFL ALYHFLPFEH NVVLGISMLA FIAVLWLTEA LHVTVTAILV PVMAVFFGIF ETQAALNNFA NSIIFLFLG GFALAAAMHH QGLDKVIADK VLAMAQGKMS VAVFMLFGVT ALLSMWISNT ATAAMMLPLV LGVLSKVDAD K QRSTYVFV LLGVAYSASI GGIATLVGSP PNAIAAAEVG LSFTDWMKFG LPTAMMMLPM AIAILYFLLK PTLNGMFELD RA PVNWDKG KVVTLGIFGL TVFLWIFSSP INAALGGFKS FDTLVALGAI LMLSFARVVH WKEIQKTADW GVLLLFGGGL CLS NVLKQT GTSVFLANAL SDMVSHMGIF VVILVVATFV VFLTEFASNT ASAALLIPVF ATVAEAFGMS PVLLSVLIAV AASC AFMLP VATPPNAIVF ASGHIKQSEM MRVGLYLNIA CIGLLTAIAM LFWQ

UniProtKB: Transporter, NadC family

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Macromolecule #2: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 2 / Number of copies: 4
Molecular weightTheoretical: 22.99 Da

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 5 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.3 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 4253 / Average exposure time: 2.34 sec. / Average electron dose: 65.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1520631
Startup modelType of model: OTHER
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.83 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 144865
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5ul9


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7t9g:
Structure of VcINDY-Na+

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