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- EMDB-42614: Structure of NaCT-PF4a complex -

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Basic information

Entry
Database: EMDB / ID: EMD-42614
TitleStructure of NaCT-PF4a complex
Map dataSharpened map of NaCT-PF4a in Ci-Ci conformation
Sample
  • Complex: Dimer of NaCT in complex with PF4a
    • Protein or peptide: Solute carrier family 13 member 5
  • Ligand: SODIUM ION
  • Ligand: (2R)-2-hydroxy-2-[2-(2-methoxy-5-methylpyridin-3-yl)ethyl]butanedioic acid
KeywordsNa(+)/citrate cotransporter(NaCT) / Solute carries / Elevator type alternating access / membrane protein / TRANSPORT PROTEIN
Function / homology
Function and homology information


oxaloacetate transport / organic acid:sodium symporter activity / fumarate transport / succinate transport / sodium:dicarboxylate symporter activity / citrate transmembrane transporter activity / citrate transport / alpha-ketoglutarate transport / Sodium-coupled sulphate, di- and tri-carboxylate transporters / succinate transmembrane transporter activity ...oxaloacetate transport / organic acid:sodium symporter activity / fumarate transport / succinate transport / sodium:dicarboxylate symporter activity / citrate transmembrane transporter activity / citrate transport / alpha-ketoglutarate transport / Sodium-coupled sulphate, di- and tri-carboxylate transporters / succinate transmembrane transporter activity / cellular response to lithium ion / transmembrane transport / nucleoplasm / identical protein binding / plasma membrane / cytosol
Similarity search - Function
Sodium/sulphate symporter, conserved site / Sodium:sulfate symporter family signature. / Sodium:sulfate symporter transmembrane region / Solute carrier family 13
Similarity search - Domain/homology
Na(+)/citrate cotransporter
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.13 Å
AuthorsLi Y / Wang DN / Mindell JA / Rice WJ / Song J / Mikusevic V / Marden JJ / Becerril A / Kuang H / Wang B
Funding support United States, 4 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 Diabetes and Digestive and Kidney Disease (NIH/NIDDK)R01DK135088 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI165782 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM121994 United States
CitationJournal: Nat Struct Mol Biol / Year: 2025
Title: Substrate translocation and inhibition in human dicarboxylate transporter NaDC3.
Authors: Yan Li / Jinmei Song / Vedrana Mikusevic / Jennifer J Marden / Alissa Becerril / Huihui Kuang / Bing Wang / William J Rice / Joseph A Mindell / Da-Neng Wang /
Abstract: The human high-affinity sodium-dicarboxylate cotransporter (NaDC3) imports various substrates into the cell as tricarboxylate acid cycle intermediates, lipid biosynthesis precursors and signaling ...The human high-affinity sodium-dicarboxylate cotransporter (NaDC3) imports various substrates into the cell as tricarboxylate acid cycle intermediates, lipid biosynthesis precursors and signaling molecules. Understanding the cellular signaling process and developing inhibitors require knowledge of the structural basis of the dicarboxylate specificity and inhibition mechanism of NaDC3. To this end, we determined the cryo-electron microscopy structures of NaDC3 in various dimers, revealing the protomer in three conformations: outward-open C, outward-occluded C and inward-open C. A dicarboxylate is first bound and recognized in C and how the substrate interacts with NaDC3 in C likely helps to further determine the substrate specificity. A phenylalanine from the scaffold domain interacts with the bound dicarboxylate in the C state and modulates the kinetic barrier to the transport domain movement. Structural comparison of an inhibitor-bound structure of NaDC3 to that of the sodium-dependent citrate transporter suggests ways for making an inhibitor that is specific for NaDC3.
History
DepositionNov 2, 2023-
Header (metadata) releaseDec 25, 2024-
Map releaseDec 25, 2024-
UpdateApr 2, 2025-
Current statusApr 2, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42614.png

Downloads & links

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Map

FileDownload / File: emd_42614.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map of NaCT-PF4a in Ci-Ci conformation
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 256 pix.
= 211.2 Å		0.83 Å/pix.
x 256 pix.
= 211.2 Å		0.83 Å/pix.
x 256 pix.
= 211.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.825 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.297
Minimum - Maximum-2.061356 - 2.966843
Average (Standard dev.)0.00089595263 (±0.059946496)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 211.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_42614_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map of NaCT-PF4a in Ci-Ci conformation

Fileemd_42614_additional_1.map
AnnotationUnsharpened map of NaCT-PF4a in Ci-Ci conformation
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A of NaCT-PF4a in Ci-Ci conformation

Fileemd_42614_half_map_1.map
AnnotationHalf map A of NaCT-PF4a in Ci-Ci conformation
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B of NaCT-PF4a in Ci-Ci conformation

Fileemd_42614_half_map_2.map
AnnotationHalf map B of NaCT-PF4a in Ci-Ci conformation
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dimer of NaCT in complex with PF4a

EntireName: Dimer of NaCT in complex with PF4a
Components
  • Complex: Dimer of NaCT in complex with PF4a
    • Protein or peptide: Solute carrier family 13 member 5
  • Ligand: SODIUM ION
  • Ligand: (2R)-2-hydroxy-2-[2-(2-methoxy-5-methylpyridin-3-yl)ethyl]butanedioic acid

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Supramolecule #1: Dimer of NaCT in complex with PF4a

SupramoleculeName: Dimer of NaCT in complex with PF4a / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 63.062 KDa

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Macromolecule #1: Solute carrier family 13 member 5

MacromoleculeName: Solute carrier family 13 member 5 / type: protein_or_peptide / ID: 1 / Details: Solute carrier family 13 member 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 63.110812 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MASALSYVSK FKSFVILFVT PLLLLPLVIL MPAKFVRCAY VIILMAIYWC TEVIPLAVTS LMPVLLFPLF QILDSRQVCV QYMKDTNML FLGGLIVAVA VERWNLHKRI ALRTLLWVGA KPARLMLGFM GVTALLSMWI SNTATTAMMV PIVEAILQQM E ATSAATEA ...String:
MASALSYVSK FKSFVILFVT PLLLLPLVIL MPAKFVRCAY VIILMAIYWC TEVIPLAVTS LMPVLLFPLF QILDSRQVCV QYMKDTNML FLGGLIVAVA VERWNLHKRI ALRTLLWVGA KPARLMLGFM GVTALLSMWI SNTATTAMMV PIVEAILQQM E ATSAATEA GLELVDKGKA KELPGSQVIF EGPTLGQQED QERKRLCKAM TLCICYAASI GGTATLTGTG PNVVLLGQMN EL FPDSKDL VNFASWFAFA FPNMLVMLLF AWLWLQFVYM RFNFKKSWGC GLESKKNEKA ALKVLQEEYR KLGPLSFAEI NVL ICFFLL VILWFSRDPG FMPGWLTVAW VEGETKYVSD ATVAIFVATL LFIVPSQKPK FNFRSQTEEE RKTPFYPPPL LDWK VTQEK VPWGIVLLLG GGFALAKGSE ASGLSVWMGK QMEPLHAVPP AAITLILSLL VAVFTECTSN VATTTLFLPI FASMS RSIG LNPLYIMLPC TLSASFAFML PVATPPNAIV FTYGHLKVAD MVKTGVIMNI IGVFCVFLAV NTWGRAIFDL DHFPDW ANV THIET

UniProtKB: Na(+)/citrate cotransporter

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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: (2R)-2-hydroxy-2-[2-(2-methoxy-5-methylpyridin-3-yl)ethyl]butaned...

MacromoleculeName: (2R)-2-hydroxy-2-[2-(2-methoxy-5-methylpyridin-3-yl)ethyl]butanedioic acid
type: ligand / ID: 3 / Number of copies: 2 / Formula: XKC
Molecular weightTheoretical: 283.277 Da

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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
Component:
ConcentrationFormulaName
25.0 mMTris-HClTris (Hydroxymethyl) Aminomethane Hydrochloride
300.0 mMNaClsodium chloride
200.0 uMPF4a(R)-2-Hydroxy-2-(2-(2-methoxy-5-methylpyridin-3-yl)ethyl)-succinic Acid (4a)
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.026000000000000002 kPa / Details: hold 10s
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281.15 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was performed an amphipol(PMAL-C8) exchange

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 80.0 K / Max: 80.0 K
Alignment procedureComa free - Residual tilt: 0.05 mrad
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 12545 / Average exposure time: 2.5 sec. / Average electron dose: 61.19 e/Å2
Details: 5516 untilted images and 7029 tilted images were collected in super resolution mode at 50 frames per micrograph
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 3.0 µm / Calibrated defocus min: 0.8 µm / Calibrated magnification: 105000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.6 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsThe micrographs with an overall resolution worse than 5 angstrom were excluded
Particle selectionNumber selected: 6132038
Details: Particles were selected from 5227 untilted images and 6942 40 degree tilted images
Startup modelType of model: OTHER / Details: Ab-initio reconstruction in cryoSPARC
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.13 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1)
Details: Non-uniform refinement and local refinement with a mask
Number images used: 745845
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1) / Details: branch-and-bound maximum likelihood
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1) / Details: branch-and-bound maximum likelihood
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC (ver. 3.3.1)
Details: Number of particles for 4 classes are 360227, 385618, 660, 72685. The reported resolutions are 3.38, 3.38, 7.67, 3.62 angstrom, respectively.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7jsk


Chain - Chain ID: AB / Chain - Residue range: 1-568 / Chain - Source name: PDB / Chain - Initial model type: experimental model / Details: The whole model was used as an initial model
DetailsInitial local fitting was done using Chimera and then coot was used for ajustment.
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 43.93 / Target criteria: cross-correlation coefficient
Output model

PDB-8uvb:
Structure of NaCT-PF4a complex

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