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- EMDB-44979: human sodium chloride cotransporter NCC S344E in the phosphorylat... -

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Basic information

Entry
Database: EMDB / ID: EMD-44979
Titlehuman sodium chloride cotransporter NCC S344E in the phosphorylation state and in complex with hydrochlorothiazide
Map datahuman sodium chloride cotransporter NCC S344E in the phosphorylation state and in complex with hydrochlorothiazide
Sample
  • Complex: Phosphorylated SLC12A3 transporter in complex with hydrochlorothiazide
    • Protein or peptide: Solute carrier family 12 member 3
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: 6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide
  • Ligand: water
Keywordssodium chloride cotransporter / phosphorylation / thiazide diuretics / TRANSPORT PROTEIN
Function / homology
Function and homology information


sodium:chloride symporter activity / apical plasma membrane
Similarity search - Function
Thiazide-sensitive Na-K-Cl co-transporter / Amino acid permease, N-terminal / Amino acid permease N-terminal / SLC12A transporter, C-terminal / Solute carrier family 12 / Amino acid permease/ SLC12A domain / SLC12A transporter family / Amino acid permease
Similarity search - Domain/homology
Solute carrier family 12 member 3
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsCao EH / Zhao YX
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)5R01DK128592 United States
CitationJournal: Nat Commun / Year: 2024
Title: Structural bases for Na-Cl cotransporter inhibition by thiazide diuretic drugs and activation by kinases.
Authors: Yongxiang Zhao / Heidi Schubert / Alan Blakely / Biff Forbush / Micholas Dean Smith / Jesse Rinehart / Erhu Cao /
Abstract: The Na-Cl cotransporter (NCC) drives salt reabsorption in the kidney and plays a decisive role in balancing electrolytes and blood pressure. Thiazide and thiazide-like diuretics inhibit NCC-mediated ...The Na-Cl cotransporter (NCC) drives salt reabsorption in the kidney and plays a decisive role in balancing electrolytes and blood pressure. Thiazide and thiazide-like diuretics inhibit NCC-mediated renal salt retention and have been cornerstones for treating hypertension and edema since the 1950s. Here we determine NCC co-structures individually complexed with the thiazide drug hydrochlorothiazide, and two thiazide-like drugs chlorthalidone and indapamide, revealing that they fit into an orthosteric site and occlude the NCC ion translocation pathway. Aberrant NCC activation by the WNKs-SPAK kinase cascade underlies Familial Hyperkalemic Hypertension, but it remains unknown whether/how phosphorylation transforms the NCC structure to accelerate ion translocation. We show that an intracellular amino-terminal motif of NCC, once phosphorylated, associates with the carboxyl-terminal domain, and together, they interact with the transmembrane domain. These interactions suggest a phosphorylation-dependent allosteric network that directly influences NCC ion translocation.
History
DepositionMay 21, 2024-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateMay 21, 2025-
Current statusMay 21, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44979.png

Downloads & links

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Map

FileDownload / File: emd_44979.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationhuman sodium chloride cotransporter NCC S344E in the phosphorylation state and in complex with hydrochlorothiazide
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 360 pix.
= 297. Å		0.83 Å/pix.
x 360 pix.
= 297. Å		0.83 Å/pix.
x 360 pix.
= 297. 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: 0.3
Minimum - Maximum-1.1848246 - 2.103523
Average (Standard dev.)-0.0009846067 (±0.05774519)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 297.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Additional Map

Fileemd_44979_additional_1.map
AnnotationAdditional Map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_44979_half_map_1.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map B

Fileemd_44979_half_map_2.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Phosphorylated SLC12A3 transporter in complex with hydrochlorothiazide

EntireName: Phosphorylated SLC12A3 transporter in complex with hydrochlorothiazide
Components
  • Complex: Phosphorylated SLC12A3 transporter in complex with hydrochlorothiazide
    • Protein or peptide: Solute carrier family 12 member 3
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: 6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide
  • Ligand: water

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Supramolecule #1: Phosphorylated SLC12A3 transporter in complex with hydrochlorothiazide

SupramoleculeName: Phosphorylated SLC12A3 transporter in complex with hydrochlorothiazide
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Solute carrier family 12 member 3

MacromoleculeName: Solute carrier family 12 member 3 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 113.40668 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAELPTTETP GDATLCSGRF TISTLLSSDE PSPPAAYDSS HPSHLTHSST FCMR(TPO)FGYN(TPO) IDVVPTYEHY AN (SEP)TQPGEP RKVRPTLADL HSFLKEGRHL HALAFDSRPS HEMTDGLVEG EAGTSSEKNP EEPVRFGWVK GVMIRCMLN IWGVILYLRL ...String:
MAELPTTETP GDATLCSGRF TISTLLSSDE PSPPAAYDSS HPSHLTHSST FCMR(TPO)FGYN(TPO) IDVVPTYEHY AN (SEP)TQPGEP RKVRPTLADL HSFLKEGRHL HALAFDSRPS HEMTDGLVEG EAGTSSEKNP EEPVRFGWVK GVMIRCMLN IWGVILYLRL PWITAQAGIV LTWIIILLSV TVTSITGLSI SAISTNGKVK SGGTYFLISR SLGPELGGSI GLIFAFANAV GVAMHTVGF AETVRDLLQE YGAPIVDPIN DIRIIAVVSV TVLLAISLAG MEWESKAQVL FFLVIMVSFA NYLVGTLIPP S EDKASKGF FSYRADIFVQ NLVPDWRGPD GTFFGMFEIF FPSATGILAG ANISGDLKDP AIAIPKGTLM AIFWTTISYL AI SATIGSC VVRDASGVLN DTVTPGWGAC EGLACSYGWN FTECTQQHSC HYGLINYYQT MSMVSGFAPL ITAGIFGATL SSA LACLVS AAKVFQCLCE DQLYPLIGFF GKGYGKNKEP VRGYLLAYAI AVAFIIIAEL NTIAPIISNF FLCSYALINF SCFH ASITN SPGWRPSFQY YNKWAALFGA IISVVIMFLL TWWAALIAIG VVLFLLLYVI YKKPEVNWGS SVQAGSYNLA LSYSV GLNE VEDHIKNYRP QCLVLTGPPN FRPALVDFVG TFTRNLSLMI CGHVLIGPHK QRMPELQLIA NGHTKWLNKR KIKAFY SDV IAEDLRRGVQ ILMQAAGLGR MKPNILVVGF KKNWQSAHPA TVEDYIGILH DAFDFNYGVC VMRMREGLNV SKMMQAH IN PVFDPAEDGK EASARVDPKA LVKEEQATTI FQSEQGKKTI DIYWLFDDGG LTLLIPYLLG RKRRWSKCKI RVFVGGQI N RMDQERKAII SLLSKFRLGF HEVHILPDIN QNPRAEHTKR FEDMIAPFRL NDGFKDEATV NEMRRDCPWK ISDEEITKN RVKSLRQVRL NEIVLDYSRD AALIVITLPI GRKGKCPSSL YMAWLETLSQ DLRPPVILIR GNQENVLTFY CQ

UniProtKB: Solute carrier family 12 member 3

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Macromolecule #2: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 2 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #3: 6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-...

MacromoleculeName: 6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide
type: ligand / ID: 3 / Number of copies: 1 / Formula: HCZ
Molecular weightTheoretical: 297.739 Da
Chemical component information

ChemComp-HCZ:
6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide / medication*YM

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 6 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
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 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 4 K

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 3281696
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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