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- EMDB-22456: Structure of the NaCT-PF2 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-22456
TitleStructure of the NaCT-PF2 complex
Map dataNaCT-PF2 complex
Sample
  • Organelle or cellular component: Dimer of NaCT in complex with PF2
    • Protein or peptide: Solute carrier family 13 member 5
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: (2R)-2-[2-(4-tert-butylphenyl)ethyl]-2-hydroxybutanedioic acid
  • Ligand: SODIUM ION
KeywordsTransporter / Inhibitor / MEMBRANE PROTEIN
Function / homology
Function and homology information


citrate transmembrane transporter activity / oxaloacetate transport / succinate transport / citrate transport / organic acid:sodium symporter activity / sodium:dicarboxylate symporter activity / fumarate transport / Sodium-coupled sulphate, di- and tri-carboxylate transporters / alpha-ketoglutarate transport / succinate transmembrane transporter activity ...citrate transmembrane transporter activity / oxaloacetate transport / succinate transport / citrate transport / organic acid:sodium symporter activity / sodium:dicarboxylate symporter activity / fumarate transport / Sodium-coupled sulphate, di- and tri-carboxylate transporters / alpha-ketoglutarate transport / succinate transmembrane transporter activity / cellular response to lithium ion / transmembrane transport / nucleoplasm / identical protein binding / plasma membrane / cytosol
Similarity search - Function
Sodium:sulfate symporter transmembrane region / Sodium/sulphate symporter, conserved site / Sodium:sulfate symporter family signature. / Solute carrier family 13
Similarity search - Domain/homology
Na(+)/citrate cotransporter
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.12 Å
AuthorsSauer DB / Wang B
Funding support United States, 7 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
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).
History
DepositionAug 14, 2020-
Header (metadata) releaseFeb 24, 2021-
Map releaseFeb 24, 2021-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 11
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 11
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7jsj
  • Surface level: 11
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22456.png

Downloads & links

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Map

FileDownload / File: emd_22456.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNaCT-PF2 complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 300 pix.
= 321.9 Å		1.07 Å/pix.
x 300 pix.
= 321.9 Å		1.07 Å/pix.
x 300 pix.
= 321.9 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.073 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 10.1 / Movie #1: 11
Minimum - Maximum-34.530887999999997 - 61.730637000000002
Average (Standard dev.)-0.000000000004667 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 321.9 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0731.0731.073
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z321.900321.900321.900
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ410410410
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-34.53161.731-0.000

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Supplemental data

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Sample components

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Entire : Dimer of NaCT in complex with PF2

EntireName: Dimer of NaCT in complex with PF2
Components
  • Organelle or cellular component: Dimer of NaCT in complex with PF2
    • Protein or peptide: Solute carrier family 13 member 5
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: (2R)-2-[2-(4-tert-butylphenyl)ethyl]-2-hydroxybutanedioic acid
  • Ligand: SODIUM ION

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Supramolecule #1: Dimer of NaCT in complex with PF2

SupramoleculeName: Dimer of NaCT in complex with PF2 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 125 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 / 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: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 2 / Number of copies: 2 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #3: (2R)-2-[2-(4-tert-butylphenyl)ethyl]-2-hydroxybutanedioic acid

MacromoleculeName: (2R)-2-[2-(4-tert-butylphenyl)ethyl]-2-hydroxybutanedioic acid
type: ligand / ID: 3 / Number of copies: 2 / Formula: X3M
Molecular weightTheoretical: 294.343 Da
Chemical component information

ChemComp-X3M:
(2R)-2-[2-(4-tert-butylphenyl)ethyl]-2-hydroxybutanedioic acid

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Macromolecule #4: SODIUM ION

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

BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average exposure time: 10.0 sec. / Average electron dose: 69.42 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.3000000000000003 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 22500
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.12 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 600496
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC

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