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- PDB-7jsk: Structure of the NaCT-Citrate complex -

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Basic information

Entry
Database: PDB / ID: 7jsk
TitleStructure of the NaCT-Citrate complex
ComponentsSolute carrier family 13 member 5
KeywordsMEMBRANE PROTEIN / Transporter
Function / homology
Function and homology information


citrate transmembrane transporter activity / citrate transport / sodium:dicarboxylate symporter activity / succinate transmembrane transporter activity / anion transmembrane transport / integral component of membrane / nucleoplasm / plasma membrane / cytosol
Sodium/sulphate symporter, conserved site / Solute carrier family 13
Solute carrier family 13 member 5
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.04 Å
AuthorsSauer, D.B. / Wang, B. / Song, J. / Rice, W.J. / Wang, D.N.
Funding support United States, 7items
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
American Cancer Society129844-PF-17-135-01-TBE United States
Department of Defense (DOD, United States)W81XWH-16-1-0153 United States
The G. Harold and Leila Y. Mathers Foundation United States
TESS Research Foundation United States
American Epilepsy Society United States
CitationJournal: Nature / Year: 2021
Title: Structure and inhibition mechanism of the human citrate transporter NaCT.
Authors: David B Sauer / Jinmei Song / Bing Wang / Jacob K Hilton / Nathan K Karpowich / Joseph A Mindell / William J Rice / Da-Neng Wang /
Abstract: Citrate is best known as an intermediate in the tricarboxylic acid cycle of the cell. In addition to this essential role in energy metabolism, the tricarboxylate anion also acts as both a precursor ...Citrate is best known as an intermediate in the tricarboxylic acid cycle of the cell. In addition to this essential role in energy metabolism, the tricarboxylate anion also acts as both a precursor and a regulator of fatty acid synthesis. Thus, the rate of fatty acid synthesis correlates directly with the cytosolic concentration of citrate. Liver cells import citrate through the sodium-dependent citrate transporter NaCT (encoded by SLC13A5) and, as a consequence, this protein is a potential target for anti-obesity drugs. Here, to understand the structural basis of its inhibition mechanism, we determined cryo-electron microscopy structures of human NaCT in complexes with citrate or a small-molecule inhibitor. These structures reveal how the inhibitor-which binds to the same site as citrate-arrests the transport cycle of NaCT. The NaCT-inhibitor structure also explains why the compound selectively inhibits NaCT over two homologous human dicarboxylate transporters, and suggests ways to further improve the affinity and selectivity. Finally, the NaCT structures provide a framework for understanding how various mutations abolish the transport activity of NaCT in the brain and thereby cause epilepsy associated with mutations in SLC13A5 in newborns (which is known as SLC13A5-epilepsy).
Validation Report
SummaryFull reportAbout validation report
History
DepositionAug 14, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 24, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 3, 2021Group: Data processing / Database references / Category: citation / citation_author / em_3d_reconstruction
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _em_3d_reconstruction.num_particles / _em_3d_reconstruction.resolution
Revision 1.2Mar 10, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Assembly

Deposited unit
A: Solute carrier family 13 member 5
B: Solute carrier family 13 member 5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)127,14010
Polymers126,2222
Non-polymers9198
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6180 Å2
ΔGint-83 kcal/mol
Surface area37750 Å2

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Components

#1: Protein Solute carrier family 13 member 5 / Na(+)/citrate cotransporter / NaCT / Sodium-coupled citrate transporter / Sodium-dependent citrate transporter


Mass: 63110.812 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC13A5, NACT / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q86YT5
#2: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#3: Chemical ChemComp-CIT / CITRIC ACID / Citric acid


Mass: 192.124 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H8O7 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-NA / SODIUM ION / Sodium


Mass: 22.990 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Na
Has ligand of interestY

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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: The NaCT-Citrate complex / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.125 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 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 FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 1700 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Image recordingAverage exposure time: 8 sec. / Electron dose: 57.71 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter slit width: 20 eV
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.18_3855: / Classification: refinement
EM software
IDNameCategory
2Leginonimage acquisition
4WarpCTF correction
7Cootmodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 563708 / Symmetry type: POINT
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0117558
ELECTRON MICROSCOPYf_angle_d1.13310308
ELECTRON MICROSCOPYf_dihedral_angle_d17.5452658
ELECTRON MICROSCOPYf_chiral_restr0.2041248
ELECTRON MICROSCOPYf_plane_restr0.0061236

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