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- EMDB-25092: Human NKCC1 state Fu-II -

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Basic information

Entry
Database: EMDB / ID: EMD-25092
TitleHuman NKCC1 state Fu-II
Map data
Sample
  • Complex: NKCC1 with furosemide
    • Protein or peptide: Solute carrier family 12 member 2
  • Ligand: 5-(AMINOSULFONYL)-4-CHLORO-2-[(2-FURYLMETHYL)AMINO]BENZOIC ACID
Keywordsfurosemide / loop diuretic / diuretic / sodium / potassium / chloride / co-transporter / CTD / NKCC1 / MEMBRANE PROTEIN
Function / homology
Function and homology information


positive regulation of cell volume / positive regulation of aspartate secretion / transepithelial ammonium transport / regulation of matrix metallopeptidase secretion / cell body membrane / inorganic anion import across plasma membrane / inorganic cation import across plasma membrane / chloride:monoatomic cation symporter activity / sodium:potassium:chloride symporter activity / metal ion transmembrane transporter activity ...positive regulation of cell volume / positive regulation of aspartate secretion / transepithelial ammonium transport / regulation of matrix metallopeptidase secretion / cell body membrane / inorganic anion import across plasma membrane / inorganic cation import across plasma membrane / chloride:monoatomic cation symporter activity / sodium:potassium:chloride symporter activity / metal ion transmembrane transporter activity / transepithelial chloride transport / potassium ion transmembrane transporter activity / Cation-coupled Chloride cotransporters / ammonium transmembrane transport / sodium ion homeostasis / intracellular chloride ion homeostasis / negative regulation of vascular wound healing / chloride ion homeostasis / cellular response to potassium ion / ammonium channel activity / intracellular potassium ion homeostasis / cell projection membrane / intracellular sodium ion homeostasis / sodium ion import across plasma membrane / potassium ion homeostasis / cellular response to chemokine / T cell chemotaxis / hyperosmotic response / gamma-aminobutyric acid signaling pathway / cell volume homeostasis / regulation of spontaneous synaptic transmission / maintenance of blood-brain barrier / potassium ion import across plasma membrane / transport across blood-brain barrier / sodium ion transmembrane transport / lateral plasma membrane / monoatomic ion transport / chloride transmembrane transport / basal plasma membrane / cell projection / cell periphery / Hsp90 protein binding / cytoplasmic vesicle membrane / extracellular vesicle / cell body / protein-folding chaperone binding / basolateral plasma membrane / neuron projection / apical plasma membrane / neuronal cell body / protein kinase binding / extracellular exosome / membrane / plasma membrane
Similarity search - Function
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
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.87 Å
AuthorsMoseng MA
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI145068 United States
CitationJournal: Sci Adv / Year: 2022
Title: Inhibition mechanism of NKCC1 involves the carboxyl terminus and long-range conformational coupling.
Authors: Mitchell A Moseng / Chih-Chia Su / Kerri Rios / Meng Cui / Meinan Lyu / Przemyslaw Glaza / Philip A Klenotic / Eric Delpire / Edward W Yu /
Abstract: The Na-K-2Cl cotransporter-1 (NKCC1) is an electroneutral Na-dependent transporter responsible for simultaneously translocating Na, K, and Cl ions into cells. In human tissue, NKCC1 plays a critical ...The Na-K-2Cl cotransporter-1 (NKCC1) is an electroneutral Na-dependent transporter responsible for simultaneously translocating Na, K, and Cl ions into cells. In human tissue, NKCC1 plays a critical role in regulating cytoplasmic volume, fluid intake, chloride homeostasis, and cell polarity. Here, we report four structures of human NKCC1 (hNKCC1), both in the absence and presence of loop diuretic (bumetanide or furosemide), using single-particle cryo-electron microscopy. These structures allow us to directly observe various novel conformations of the hNKCC1 dimer. They also reveal two drug-binding sites located at the transmembrane and cytosolic carboxyl-terminal domains, respectively. Together, our findings enable us to delineate an inhibition mechanism that involves a coupled movement between the cytosolic and transmembrane domains of hNKCC1.
History
DepositionOct 4, 2021-
Header (metadata) releaseSep 28, 2022-
Map releaseSep 28, 2022-
UpdateMay 31, 2023-
Current statusMay 31, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25092.png

Downloads & links

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Map

FileDownload / File: emd_25092.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-1.6188701 - 3.1894214
Average (Standard dev.)-0.00057859166 (±0.04360365)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 428.00003 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_25092_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

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Half map: #2

Fileemd_25092_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_25092_half_map_2.map
Projections & Slices
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Sample components

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Entire : NKCC1 with furosemide

EntireName: NKCC1 with furosemide
Components
  • Complex: NKCC1 with furosemide
    • Protein or peptide: Solute carrier family 12 member 2
  • Ligand: 5-(AMINOSULFONYL)-4-CHLORO-2-[(2-FURYLMETHYL)AMINO]BENZOIC ACID

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Supramolecule #1: NKCC1 with furosemide

SupramoleculeName: NKCC1 with furosemide / 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 2

MacromoleculeName: Solute carrier family 12 member 2 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 100.435695 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GWIKGVLVRC MLNIWGVMLF IRLSWIVGQA GIGLSVLVIM MATVVTTITG LSTSAIATNG FVRGGGAYYL ISRSLGPEFG GAIGLIFAF ANAVAVAMYV VGFAETVVEL LKEHSILMID EINDIRIIGA ITVVILLGIS VAGMEWEAKA QIVLLVILLL A IGDFVIGT ...String:
GWIKGVLVRC MLNIWGVMLF IRLSWIVGQA GIGLSVLVIM MATVVTTITG LSTSAIATNG FVRGGGAYYL ISRSLGPEFG GAIGLIFAF ANAVAVAMYV VGFAETVVEL LKEHSILMID EINDIRIIGA ITVVILLGIS VAGMEWEAKA QIVLLVILLL A IGDFVIGT FIPLESKKPK GFFGYKSEIF NENFGPDFRE EETFFSVFAI FFPAATGILA GANISGDLAD PQSAIPKGTL LA ILITTLV YVGIAVSVGS CVVRDATGNV NDTIVTELTN CTSAACKLNF DFSSCESSPC SYGLMNNFQV MSMVSGFTPL ISA GIFSAT LSSALASLVS APKIFQALCK DNIYPAFQMF AKGYGKNNEP LRGYILTFLI ALGFILIAEL NVIAPIISNF FLAS YALIN FSVFHASLAK SPGWRPAFKY YNMWISLLGA ILCCIVMFVI NWWAALLTYV IVLGLYIYVT YKKPDVNWGS STQAL TYLN ALQHSIRLSG VEDHVKNFRP QCLVMTGAPN SRPALLHLVH DFTKNVGLMI CGHVHMGPRR QAMKEMSIDQ AKYQRW LIK NKMKAFYAPV HADDLREGAQ YLMQAAGLGR MKPNTLVLGF KKDWLQADMR DVDMYINLFH DAFDIQYGVV VIRLKEG L(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)KQGKN TIDVWWLFDD GGLTL LIPY LLTTKKKWKD CKIRVFIGGK INRIDHDRRA MATLLSKFRI DFSDIMVLGD INTKPKKENI IAFEEIIEPY RLHEDD KEQ DIADKMKEDE PWRITDNELE LYKTKTYRQI RLNELLKEHS STANIIVMSL PVARKGAVSS ALYMAWLEAL SKDLPPI LL VRGNHQSVLT F

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Macromolecule #2: 5-(AMINOSULFONYL)-4-CHLORO-2-[(2-FURYLMETHYL)AMINO]BENZOIC ACID

MacromoleculeName: 5-(AMINOSULFONYL)-4-CHLORO-2-[(2-FURYLMETHYL)AMINO]BENZOIC ACID
type: ligand / ID: 2 / Number of copies: 2 / Formula: FUN
Molecular weightTheoretical: 330.744 Da
Chemical component information

ChemComp-FUN:
5-(AMINOSULFONYL)-4-CHLORO-2-[(2-FURYLMETHYL)AMINO]BENZOIC ACID / medication*YM / Furosemide

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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

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 41.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.87 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 261000
FSC plot (resolution estimation)

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