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- EMDB-29096: Cryo-EM structure of human NCC (class 2) -

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Basic information

Entry
Database: EMDB / ID: EMD-29096
TitleCryo-EM structure of human NCC (class 2)
Map data
Sample
  • Complex: NCC-polythiazide complex
    • Protein or peptide: Solute carrier family 12 member 2,Solute carrier family 12 member 3 chimera
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: Polythiazide
Function / homology
Function and homology information


swim bladder inflation / Cation-coupled Chloride cotransporters / Defective SLC12A3 causes Gitelman syndrome (GS) / sodium:chloride symporter activity / chloride:monoatomic cation symporter activity / sodium:potassium:chloride symporter activity / cellular response to inorganic substance / Cation-coupled Chloride cotransporters / sodium ion homeostasis / renal sodium ion absorption ...swim bladder inflation / Cation-coupled Chloride cotransporters / Defective SLC12A3 causes Gitelman syndrome (GS) / sodium:chloride symporter activity / chloride:monoatomic cation symporter activity / sodium:potassium:chloride symporter activity / cellular response to inorganic substance / Cation-coupled Chloride cotransporters / sodium ion homeostasis / renal sodium ion absorption / chloride ion homeostasis / ear development / ammonium transmembrane transporter activity / potassium ion homeostasis / cell volume homeostasis / inner ear morphogenesis / response to aldosterone / sodium ion transport / potassium ion import across plasma membrane / response to dietary excess / sodium ion transmembrane transport / monoatomic ion transport / chloride transmembrane transport / basolateral plasma membrane / apical plasma membrane / signal transduction / extracellular exosome / ATP binding / membrane / identical protein binding / plasma membrane / cytosol
Similarity search - Function
Thiazide-sensitive Na-K-Cl co-transporter / Solute carrier family 12 member 1/2 / Solute carrier family 12 member 2 / Amino acid permease, N-terminal / Amino acid permease N-terminal / SLC12A transporter family / SLC12A transporter, C-terminal / Solute carrier family 12 / Amino acid permease/ SLC12A domain / Amino acid permease
Similarity search - Domain/homology
Solute carrier family 12 member 2 / Solute carrier family 12 member 3
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsZhang J / Fan M / Feng L
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nature / Year: 2023
Title: Structure and thiazide inhibition mechanism of the human Na-Cl cotransporter.
Authors: Minrui Fan / Jianxiu Zhang / Chien-Ling Lee / Jinru Zhang / Liang Feng /
Abstract: The sodium-chloride cotransporter (NCC) is critical for kidney physiology. The NCC has a major role in salt reabsorption in the distal convoluted tubule of the nephron, and mutations in the NCC cause ...The sodium-chloride cotransporter (NCC) is critical for kidney physiology. The NCC has a major role in salt reabsorption in the distal convoluted tubule of the nephron, and mutations in the NCC cause the salt-wasting disease Gitelman syndrome. As a key player in salt handling, the NCC regulates blood pressure and is the target of thiazide diuretics, which have been widely prescribed as first-line medications to treat hypertension for more than 60 years. Here we determined the structures of human NCC alone and in complex with a commonly used thiazide diuretic using cryo-electron microscopy. These structures, together with functional studies, reveal major conformational states of the NCC and an intriguing regulatory mechanism. They also illuminate how thiazide diuretics specifically interact with the NCC and inhibit its transport function. Our results provide critical insights for understanding the Na-Cl cotransport mechanism of the NCC, and they establish a framework for future drug design and for interpreting disease-related mutations.
History
DepositionDec 14, 2022-
Header (metadata) releaseFeb 15, 2023-
Map releaseFeb 15, 2023-
UpdateMar 8, 2023-
Current statusMar 8, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29096.png

Downloads & links

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Map

FileDownload / File: emd_29096.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.86 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-1.1591609 - 1.7261196
Average (Standard dev.)-0.0010560395 (±0.051428683)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 275.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_29096_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_29096_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_29096_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : NCC-polythiazide complex

EntireName: NCC-polythiazide complex
Components
  • Complex: NCC-polythiazide complex
    • Protein or peptide: Solute carrier family 12 member 2,Solute carrier family 12 member 3 chimera
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: Polythiazide

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Supramolecule #1: NCC-polythiazide complex

SupramoleculeName: NCC-polythiazide complex / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Solute carrier family 12 member 2,Solute carrier family 12 member...

MacromoleculeName: Solute carrier family 12 member 2,Solute carrier family 12 member 3 chimera
type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 120.372164 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSASPPISAG DYLSAPEPDA LKPAGPTPSQ SRFQVDLVTE SAGDGETTVG FDSSPPEYVA EPPPDGLRDS VSGGEKAKGR FRVVNFAAS SPDAAPAETA QNGDTVMSEG SLHSSTGGQQ HHHYDTHTNT YYLRTFGHNT IDAVPKIDFY RQTAAPLGEK L IRPTLSEL ...String:
MSASPPISAG DYLSAPEPDA LKPAGPTPSQ SRFQVDLVTE SAGDGETTVG FDSSPPEYVA EPPPDGLRDS VSGGEKAKGR FRVVNFAAS SPDAAPAETA QNGDTVMSEG SLHSSTGGQQ HHHYDTHTNT YYLRTFGHNT IDAVPKIDFY RQTAAPLGEK L IRPTLSEL HDELDKEPFE DGFANGEELT PAEESAAKDV SESKEPVRFG WVKGVMIRCM LNIWGVILYL RLPWITAQAG IV LTWIIIL LSVTVTSITG LSISAISTNG KVKSGGTYFL ISRSLGPELG GSIGLIFAFA NAVGVAMHTV GFAATVRDLL QEY GAPIVD PINDIRIIGV VSVTVLLAIS LAGMEWESKA QVLFFLVIMV SFANYLVGTL IPPSEDKASK GFFSYRADIF VQNL VPDWR GPDGTFFGMF SIFFPSATGI LAGANISGDL KDPAIAIPKG TLMAIFWTTI SYLAISATIG SCVVRDASGV LNDTV TPGW GACEGLACSY GWNFTECTQQ HSCHYGLINY YQTMSMVSGF APLITAGIFG ATLSSALACL VSAAKVFQCL CEDQLY PLI GFFGKGYGKN KEPVRGYLLA YAIAVAFIII AELNTIAPII SNFFLCSYAL INFSCFHASI TNSPGWRPSF QYYNKWA AL FGAIISVVIM FLLTWWAALI AIGVVLFLLL YVIYKKPEVN WGSSVQAGSY NLALSYSVGL NEVEDHIKNY RPQCLVLT G PPNFRPALVD FVGTFTRNLS LMICGHVLIG PHKQRMPELQ LIANGHTKWL NKRKIKAFYS DVIAEDLRRG VQILMQAAG LGRMKPNILV VGFKKNWQSA HPATVEDYIG ILHDAFDFNY GVCVMRMREG LNVSKMMQAH INPVFDPAED GKEASARVDP KALVKEEQA TTIFQSEQGK KTIDIYWLFD DGGLTLLIPY LLGRKRRWSK CKIRVFVGGQ INRMDQERKA IISLLSKFRL G FHEVHILP DINQNPRAEH TKRFEDMIAP FRLNDGFKDE ATVNEMRRDC PWKISDEEIT KNRVKSLRQV RLNEIVLDYS RD AALIVIT LPIGRKGKCP SSLYMAWLET LSQDLRPPVI LIRGNQENVL TFYCQ

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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 / Adenosine triphosphate

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Macromolecule #3: Polythiazide

MacromoleculeName: Polythiazide / type: ligand / ID: 3 / Number of copies: 2 / Formula: XZF
Molecular weightTheoretical: 439.882 Da
Chemical component information

ChemComp-XZF:
Polythiazide

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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

Concentration12 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 132256
FSC plot (resolution estimation)

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