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- EMDB-42617: Structure of NaDC3-Succinate complex in Coo-Ci conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-42617
TitleStructure of NaDC3-Succinate complex in Coo-Ci conformation
Map dataSharpened map of NaDC3-succinate in Coo-Ci conformation
Sample
  • Complex: Dimer of NaDC3 in complex with succinate
    • Protein or peptide: Na(+)/dicarboxylate cotransporter 3
  • Ligand: SODIUM ION
  • Ligand: SUCCINIC ACID
  • Ligand: TETRADECANE
  • Ligand: CHOLESTEROL
  • Ligand: 1,2-Distearoyl-sn-glycerophosphoethanolamine
KeywordsNa(+)/dicarboxylate cotransporter(NaDC3) / Solute carries / Elevator type alternating access / membrane protein / TRANSPORT PROTEIN
Function / homology
Function and homology information


dicarboxylic acid transmembrane transporter activity / dicarboxylic acid transport / high-affinity sodium:dicarboxylate symporter activity / citrate transmembrane transporter activity / citrate transport / sodium:dicarboxylate symporter activity / alpha-ketoglutarate transmembrane transporter activity / Sodium-coupled sulphate, di- and tri-carboxylate transporters / succinate transmembrane transport / succinate transmembrane transporter activity ...dicarboxylic acid transmembrane transporter activity / dicarboxylic acid transport / high-affinity sodium:dicarboxylate symporter activity / citrate transmembrane transporter activity / citrate transport / sodium:dicarboxylate symporter activity / alpha-ketoglutarate transmembrane transporter activity / Sodium-coupled sulphate, di- and tri-carboxylate transporters / succinate transmembrane transport / succinate transmembrane transporter activity / glutathione transmembrane transport / glutathione transmembrane transporter activity / lipid transport / transport across blood-brain barrier / basolateral plasma membrane / extracellular exosome / plasma membrane
Similarity search - Function
Sodium:sulfate symporter transmembrane region / Solute carrier family 13
Similarity search - Domain/homology
Na(+)/dicarboxylate cotransporter 3
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 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: 2024
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-
UpdateDec 25, 2024-
Current statusDec 25, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42617.png

Downloads & links

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Map

FileDownload / File: emd_42617.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map of NaDC3-succinate in Coo-Ci conformation
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 320 pix.
= 264. Å		0.83 Å/pix.
x 320 pix.
= 264. Å		0.83 Å/pix.
x 320 pix.
= 264. 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: 0.34
Minimum - Maximum-1.3981004 - 2.481352
Average (Standard dev.)-0.0013896363 (±0.058658753)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 264.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_42617_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 NaDC3-succinate in Coo-Ci conformation

Fileemd_42617_additional_1.map
AnnotationUnsharpened map of NaDC3-succinate in Coo-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 NaDC3-succinate in Coo-Ci conformation

Fileemd_42617_half_map_1.map
AnnotationHalf map A of NaDC3-succinate in Coo-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 NaDC3-succinate in Coo-Ci conformation

Fileemd_42617_half_map_2.map
AnnotationHalf map B of NaDC3-succinate in Coo-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 NaDC3 in complex with succinate

EntireName: Dimer of NaDC3 in complex with succinate
Components
  • Complex: Dimer of NaDC3 in complex with succinate
    • Protein or peptide: Na(+)/dicarboxylate cotransporter 3
  • Ligand: SODIUM ION
  • Ligand: SUCCINIC ACID
  • Ligand: TETRADECANE
  • Ligand: CHOLESTEROL
  • Ligand: 1,2-Distearoyl-sn-glycerophosphoethanolamine

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Supramolecule #1: Dimer of NaDC3 in complex with succinate

SupramoleculeName: Dimer of NaDC3 in complex with succinate / 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: Na(+)/dicarboxylate cotransporter 3

MacromoleculeName: Na(+)/dicarboxylate cotransporter 3 / type: protein_or_peptide / ID: 1 / Details: Solute carrier family 13 member 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 67.236023 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MAALAAAAKK VWSARRLLVL LFTPLALLPV VFALPPKEGR CLFVILLMAV YWCTEALPLS VTALLPIVLF PFMGILPSNK VCPQYFLDT NFLFLSGLIM ASAIEEWNLH RRIALKILML VGVQPARLIL GMMVTTSFLS MWLSNTASTA MMLPIANAIL K SLFGQKEV ...String:
MAALAAAAKK VWSARRLLVL LFTPLALLPV VFALPPKEGR CLFVILLMAV YWCTEALPLS VTALLPIVLF PFMGILPSNK VCPQYFLDT NFLFLSGLIM ASAIEEWNLH RRIALKILML VGVQPARLIL GMMVTTSFLS MWLSNTASTA MMLPIANAIL K SLFGQKEV RKDPSQESEE NTAAVRRNGL HTVPTEMQFL ASTEAKDHPG ETEVPLDLPA DSRKEDEYRR NIWKGFLISI PY SASIGGT ATLTGTAPNL ILLGQLKSFF PQCDVVNFGS WFIFAFPLML LFLLAGWLWI SFLYGGLSFR GWRKNKSEIR TNA EDRARA VIREEYQNLG PIKFAEQAVF ILFCMFAILL FTRDPKFIPG WASLFNPGFL SDAVTGVAIV TILFFFPSQR PSLK WWFDF KAPNTETEPL LTWKKAQETV PWNIILLLGG GFAMAKGCEE SGLSVWIGGQ LHPLENVPPA LAVLLITVVI AFFTE FASN TATIIIFLPV LAELAIRLRV HPLYLMIPGT VGCSFAFMLP VSTPPNSIAF ASGHLLVKDM VRTGLLMNLM GVLLLS LAM NTWAQTIFQL GTFPDWADMY SVNVTALPPT LANDTFRTLS GAGA

UniProtKB: Na(+)/dicarboxylate cotransporter 3

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Macromolecule #2: SODIUM ION

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

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Macromolecule #3: SUCCINIC ACID

MacromoleculeName: SUCCINIC ACID / type: ligand / ID: 3 / Number of copies: 2 / Formula: SIN
Molecular weightTheoretical: 118.088 Da
Chemical component information

ChemComp-SIN:
SUCCINIC ACID

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Macromolecule #4: TETRADECANE

MacromoleculeName: TETRADECANE / type: ligand / ID: 4 / Number of copies: 6 / Formula: C14
Molecular weightTheoretical: 198.388 Da
Chemical component information

ChemComp-C14:
TETRADECANE

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Macromolecule #5: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 5 / Number of copies: 10 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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Macromolecule #6: 1,2-Distearoyl-sn-glycerophosphoethanolamine

MacromoleculeName: 1,2-Distearoyl-sn-glycerophosphoethanolamine / type: ligand / ID: 6 / Number of copies: 2 / Formula: 3PE
Molecular weightTheoretical: 748.065 Da
Chemical component information

ChemComp-3PE:
1,2-Distearoyl-sn-glycerophosphoethanolamine / phospholipid*YM

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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

Concentration6.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMTris-HClTris (Hydroxymethyl) Aminomethane Hydrochloride
100.0 mMNaClsodium chloride
20.0 mMsuccinatesuccinate
0.02 %GDNglyco-diosgenin
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 before glow discharge
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281.15 K / Instrument: FEI VITROBOT MARK IV

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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: 5179 / Average exposure time: 1.8 sec. / Average electron dose: 52.58 e/Å2
Details: 5179 untilted images were collected in super resolution mode at 40 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: 2932803 / Details: Particles were selected from 5040 untilted images
Startup modelType of model: OTHER / Details: Ab-initio reconstruction in cryoSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 228347
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 3 classes are 10828, 202740, 228347. The reported resolutions are 8.15, 4.22, 4.22 angstom, respectively.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Chain ID: AB / Chain - Residue range: 1-602 / Chain - Source name: AlphaFold / Chain - Initial model type: in silico 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: 68.84 / Target criteria: cross-correlation coefficient
Output model

PDB-8uve:
Structure of NaDC3-Succinate complex in Coo-Ci conformation

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