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- EMDB-43411: Phosphorylated human NCC in complex with indapamide -

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Basic information

Entry
Database: EMDB / ID: EMD-43411
TitlePhosphorylated human NCC in complex with indapamide
Map dataPhosphorylated human NCC in complex with indapamide
Sample
  • Complex: Phosphorylated human NCC in complex with Indapamide
    • Protein or peptide: Solute carrier family 12 member 3
    • Protein or peptide: Solute carrier family 12 member 3
  • Ligand: 4-chloro-N-[(2S)-2-methyl-2,3-dihydro-1H-indol-1-yl]-3-sulfamoylbenzamide
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: water
KeywordsPhosphorylation / a thiazide-like drug / Indapamide / Outward-open / 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: 2.7 Å
AuthorsZhao YX / Cao EH
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)DK128592 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
DepositionJan 16, 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_43411.png

Downloads & links

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Map

FileDownload / File: emd_43411.map.gz / Format: CCP4 / Size: 209.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPhosphorylated human NCC in complex with indapamide
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 380 pix.
= 326.8 Å		0.86 Å/pix.
x 380 pix.
= 326.8 Å		0.86 Å/pix.
x 380 pix.
= 326.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.22
Minimum - Maximum-0.9438953 - 1.424901
Average (Standard dev.)0.00012993797 (±0.036809124)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions380380380
Spacing380380380
CellA=B=C: 326.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Additional Map

Fileemd_43411_additional_1.map
AnnotationAdditional Map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_43411_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_43411_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 human NCC in complex with Indapamide

EntireName: Phosphorylated human NCC in complex with Indapamide
Components
  • Complex: Phosphorylated human NCC in complex with Indapamide
    • Protein or peptide: Solute carrier family 12 member 3
    • Protein or peptide: Solute carrier family 12 member 3
  • Ligand: 4-chloro-N-[(2S)-2-methyl-2,3-dihydro-1H-indol-1-yl]-3-sulfamoylbenzamide
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: water

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Supramolecule #1: Phosphorylated human NCC in complex with Indapamide

SupramoleculeName: Phosphorylated human NCC in complex with Indapamide / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
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: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 113.364648 KDa
Recombinant expressionOrganism: Mammalian expression vector BsrGI-MCS-pcDNA3.1 (others)
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 GTFFGMFSIF 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: Solute carrier family 12 member 3

MacromoleculeName: Solute carrier family 12 member 3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 113.124711 KDa
Recombinant expressionOrganism: Mammalian expression vector BsrGI-MCS-pcDNA3.1 (others)
SequenceString: MAELPTTETP GDATLCSGRF TISTLLSSDE PSPPAAYDSS HPSHLTHSST FCMRTFGYNT IDVVPTYEHY ANSTQPGEPR KVRPTLADL HSFLKEGRHL HALAFDSRPS HEMTDGLVEG EAGTSSEKNP EEPVRFGWVK GVMIRCMLNI WGVILYLRLP W ITAQAGIV ...String:
MAELPTTETP GDATLCSGRF TISTLLSSDE PSPPAAYDSS HPSHLTHSST FCMRTFGYNT IDVVPTYEHY ANSTQPGEPR KVRPTLADL HSFLKEGRHL HALAFDSRPS HEMTDGLVEG EAGTSSEKNP EEPVRFGWVK GVMIRCMLNI WGVILYLRLP W ITAQAGIV LTWIIILLSV TVTSITGLSI SAISTNGKVK SGGTYFLISR SLGPELGGSI GLIFAFANAV GVAMHTVGFA ET VRDLLQE YGAPIVDPIN DIRIIAVVSV TVLLAISLAG MEWESKAQVL FFLVIMVSFA NYLVGTLIPP SEDKASKGFF SYR ADIFVQ NLVPDWRGPD GTFFGMFSIF FPSATGILAG ANISGDLKDP AIAIPKGTLM AIFWTTISYL AISATIGSCV VRDA SGVLN DTVTPGWGAC EGLACSYGWN FTECTQQHSC HYGLINYYQT MSMVSGFAPL ITAGIFGATL SSALACLVSA AKVFQ CLCE DQLYPLIGFF GKGYGKNKEP VRGYLLAYAI AVAFIIIAEL NTIAPIISNF FLCSYALINF SCFHASITNS PGWRPS FQY YNKWAALFGA IISVVIMFLL TWWAALIAIG VVLFLLLYVI YKKPEVNWGS SVQAGSYNLA LSYSVGLNEV EDHIKNY RP QCLVLTGPPN FRPALVDFVG TFTRNLSLMI CGHVLIGPHK QRMPELQLIA NGHTKWLNKR KIKAFYSDVI AEDLRRGV Q ILMQAAGLGR MKPNILVVGF KKNWQSAHPA TVEDYIGILH DAFDFNYGVC VMRMREGLNV SKMMQAHINP VFDPAEDGK EASARVDPKA LVKEEQATTI FQSEQGKKTI DIYWLFDDGG LTLLIPYLLG RKRRWSKCKI RVFVGGQINR MDQERKAIIS LLSKFRLGF HEVHILPDIN QNPRAEHTKR FEDMIAPFRL NDGFKDEATV NEMRRDCPWK ISDEEITKNR VKSLRQVRLN E IVLDYSRD AALIVITLPI GRKGKCPSSL YMAWLETLSQ DLRPPVILIR GNQENVLTFY CQ

UniProtKB: Solute carrier family 12 member 3

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Macromolecule #3: 4-chloro-N-[(2S)-2-methyl-2,3-dihydro-1H-indol-1-yl]-3-sulfamoylb...

MacromoleculeName: 4-chloro-N-[(2S)-2-methyl-2,3-dihydro-1H-indol-1-yl]-3-sulfamoylbenzamide
type: ligand / ID: 3 / Number of copies: 1 / Formula: BL1
Molecular weightTheoretical: 365.835 Da
Chemical component information

ChemComp-BL1:
4-chloro-N-[(2S)-2-methyl-2,3-dihydro-1H-indol-1-yl]-3-sulfamoylbenzamide / inhibitor*YM

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Macromolecule #4: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / 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 #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 5 / 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

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 40.0 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: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 2148162
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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