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- EMDB-40759: Cryo-EM structure of NKCC1 Fu_CTD -

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Basic information

Entry
Database: EMDB / ID: EMD-40759
TitleCryo-EM structure of NKCC1 Fu_CTD
Map data
Sample
  • Complex: NKCC1_Fu_CTD
    • Protein or peptide: Solute carrier family 12 member 2
  • Ligand: 5-(AMINOSULFONYL)-4-CHLORO-2-[(2-FURYLMETHYL)AMINO]BENZOIC ACID
Keywordscotransporter / dimer / ion / membrane protein / TRANSPORT PROTEIN
Function / homology
Function and homology information


chloride:monoatomic cation symporter activity / sodium ion transport / 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: 3.34 Å
AuthorsMoseng MA
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)RES224174 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
DepositionMay 10, 2023-
Header (metadata) releaseMay 17, 2023-
Map releaseMay 17, 2023-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40759.png

Downloads & links

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Map

FileDownload / File: emd_40759.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: 0.3
Minimum - Maximum-2.3896573 - 4.0222497
Average (Standard dev.)-0.000008339383 (±0.06319005)
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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Half map: #2

Fileemd_40759_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_40759_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 : NKCC1_Fu_CTD

EntireName: NKCC1_Fu_CTD
Components
  • Complex: NKCC1_Fu_CTD
    • 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_Fu_CTD

SupramoleculeName: NKCC1_Fu_CTD / 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: 45.190438 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: SSTQALTYLN ALQHSIRLSG VEDHVKNFRP QCLVMTGAPN SRPALLHLVH DFTKNVGLMI CGHVHMGPRR QAMKEMSIDQ AKYQRWLIK NKMKAFYAPV HADDLREGAQ YLMQAAGLGR MKPNTLVLGF KKDWLQADMR DVDMYINLFH DAFDIQYGVV V IRLKEGLD ...String:
SSTQALTYLN ALQHSIRLSG VEDHVKNFRP QCLVMTGAPN SRPALLHLVH DFTKNVGLMI CGHVHMGPRR QAMKEMSIDQ AKYQRWLIK NKMKAFYAPV HADDLREGAQ YLMQAAGLGR MKPNTLVLGF KKDWLQADMR DVDMYINLFH DAFDIQYGVV V IRLKEGLD ISHLQGQESK GPIVPLNVAD QKLLEASTQF QKKQGKNTID VWWLFDDGGL TLLIPYLLTT KKKWKDCKIR VF IGGKINR IDHDRRAMAT LLSKFRIDFS DIMVLGDINT KPKKENIIAF EEIIEPYRLH EDDKEQDIAD KMKEDEPWRI TDN ELELYK TKTYRQIRLN ELLKEHSSTA NIIVMSLPVA RKGAVSSALY MAWLEALSKD LPPILLVRGN HQSVLTF

UniProtKB: Solute carrier family 12 member 2

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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

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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
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: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.34 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 304440
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
FSC plot (resolution estimation)

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