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- EMDB-24074: CryoEM structure of human NKCC1 -

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Basic information

Entry
Database: EMDB / ID: EMD-24074
TitleCryoEM structure of human NKCC1
Map data
Sample
  • Complex: Full-length human NKCC1 with NaCl and KCl
    • Protein or peptide: Solute carrier family 12 member 2
  • Ligand: CHLORIDE ION
  • Ligand: POTASSIUM ION
KeywordsSodium / potassium / chloride / co-transporter / ions / human / CTD / TMD / full-length / 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 / metal ion transmembrane transporter activity / 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 ...positive regulation of cell volume / positive regulation of aspartate secretion / transepithelial ammonium transport / regulation of matrix metallopeptidase secretion / metal ion transmembrane transporter activity / 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 / 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 / cell projection membrane / ammonium channel activity / intracellular potassium ion homeostasis / potassium ion homeostasis / sodium ion import across plasma membrane / cellular response to chemokine / T cell chemotaxis / intracellular sodium ion homeostasis / hyperosmotic response / cell volume homeostasis / gamma-aminobutyric acid signaling pathway / 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 / protein-folding chaperone binding / cell body / 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) / human (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.47 Å
AuthorsMoseng MA
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)RO1 AI145069 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 19, 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_24074.png

Downloads & links

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Map

FileDownload / File: emd_24074.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.17
Minimum - Maximum-1.3612102 - 1.9308629
Average (Standard dev.)-0.00054561585 (±0.025451398)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 388.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_24074_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_24074_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_24074_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Full-length human NKCC1 with NaCl and KCl

EntireName: Full-length human NKCC1 with NaCl and KCl
Components
  • Complex: Full-length human NKCC1 with NaCl and KCl
    • Protein or peptide: Solute carrier family 12 member 2
  • Ligand: CHLORIDE ION
  • Ligand: POTASSIUM ION

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Supramolecule #1: Full-length human NKCC1 with NaCl and KCl

SupramoleculeName: Full-length human NKCC1 with NaCl and KCl / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 131 KDa

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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: human (human)
Molecular weightTheoretical: 102.518414 KDa
SequenceString: KFGWIKGVLV RCMLNIWGVM LFIRLSWIVG QAGIGLSVLV IMMATVVTTI TGLSTSAIAT NGFVRGGGAY YLISRSLGPE FGGAIGLIF AFANAVAVAM YVVGFAETVV ELLKEHSILM IDEINDIRII GAITVVILLG ISVAGMEWEA KAQIVLLVIL L LAIGDFVI ...String:
KFGWIKGVLV RCMLNIWGVM LFIRLSWIVG QAGIGLSVLV IMMATVVTTI TGLSTSAIAT NGFVRGGGAY YLISRSLGPE FGGAIGLIF AFANAVAVAM YVVGFAETVV ELLKEHSILM IDEINDIRII GAITVVILLG ISVAGMEWEA KAQIVLLVIL L LAIGDFVI GTFIPLESKK PKGFFGYKSE IFNENFGPDF REEETFFSVF AIFFPAATGI LAGANISGDL ADPQSAIPKG TL LAILITT LVYVGIAVSV GSCVVRDATG NVNDTIVTEL TNCTSAACKL NFDFSSCESS PCSYGLMNNF QVMSMVSGFT PLI SAGIFS ATLSSALASL VSAPKIFQAL CKDNIYPAFQ MFAKGYGKNN EPLRGYILTF LIALGFILIA ELNVIAPIIS NFFL ASYAL INFSVFHASL AKSPGWRPAF KYYNMWISLL GAILCCIVMF VINWWAALLT YVIVLGLYIY VTYKKPDVNW GSSTQ ALTY LNALQHSIRL SGVEDHVKNF RPQCLVMTGA PNSRPALLHL VHDFTKNVGL MICGHVHMGP RRQAMKEMSI DQAKYQ RWL IKNKMKAFYA PVHADDLREG AQYLMQAAGL GRMKPNTLVL GFKKDWLQAD MRDVDMYINL FHDAFDIQYG VVVIRLK EG LDISHLQGQE ELLSSQEKSP GTKDVVVSVE YSKKSDLDTS KPLSEKPITH KVEEEDGKTA TQPLLKKESK GPIVPLNV A DQKLLEASTQ FQQKQGKNTI DVWWLFDDGG LTLLIPYLLT TKKKWKDCKI RVFIGGKINR IDHDRRAMAT LLSKFRIDF SDIMVLGDIN TKPKKENIIA FEEIIEPYRL HEDDKEQDIA DKMKEDEPWR ITDNELELYK TKTYRQIRLN ELLKEHSSTA NIIVMSLPV ARKGAVSSAL YMAWLEALSK DLPPILLVRG NHQS

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Macromolecule #2: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 2 / Number of copies: 2 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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Macromolecule #3: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: K
Molecular weightTheoretical: 39.098 Da

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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
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300
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: 3.47 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 354623
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
FSC plot (resolution estimation)

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