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- EMDB-24812: Cryo-EM structure of human NKCC1 K289NA492E bound with bumetanide -

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ID or keywords:

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Basic information

Entry
Database: EMDB / ID: EMD-24812
TitleCryo-EM structure of human NKCC1 K289NA492E bound with bumetanide
Map data
Sample
  • Complex: hNKCC1 K289NA492E bound with bumetanide
    • Protein or peptide: Solute carrier family 12 member 2
  • Ligand: POTASSIUM IONPotassium
  • Ligand: CHLORIDE IONChloride
  • Ligand: 3-(butylamino)-4-phenoxy-5-sulfamoylbenzoic acid
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 transmembrane transporter activity / intracellular potassium ion homeostasis / cell projection membrane / sodium ion import across plasma membrane / intracellular sodium ion homeostasis / 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 / cytoplasmic vesicle membrane / Hsp90 protein binding / 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: 3.6 Å
AuthorsZhao YX / Cao E
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2022
Title: Structural basis for inhibition of the Cation-chloride cotransporter NKCC1 by the diuretic drug bumetanide
Authors: Zhao Y / Roy K / Vidossich P / Cancedda L / De Vivo M / Forbush B / Cao E
History
DepositionSep 2, 2021-
Header (metadata) releaseMay 25, 2022-
Map releaseMay 25, 2022-
UpdateMay 25, 2022-
Current statusMay 25, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_24812.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.6
Minimum - Maximum-2.3452425 - 3.2074893
Average (Standard dev.)0.008169603 (±0.073862545)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 271.36 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_24812_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : hNKCC1 K289NA492E bound with bumetanide

EntireName: hNKCC1 K289NA492E bound with bumetanide
Components
  • Complex: hNKCC1 K289NA492E bound with bumetanide
    • Protein or peptide: Solute carrier family 12 member 2
  • Ligand: POTASSIUM IONPotassium
  • Ligand: CHLORIDE IONChloride
  • Ligand: 3-(butylamino)-4-phenoxy-5-sulfamoylbenzoic acid

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Supramolecule #1: hNKCC1 K289NA492E bound with bumetanide

SupramoleculeName: hNKCC1 K289NA492E bound with bumetanide / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Mammalian expression vector BsrGI-MCS-pcDNA3.1 (others)

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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: 131.841594 KDa
Recombinant expressionOrganism: Mammalian expression vector BsrGI-MCS-pcDNA3.1 (others)
SequenceString: GAMGSEPRPT APSSGAPGLA GVGETPSAAA LAAARVELPG TAVPSVPEDA APASRDGGGV RDEGPAAAGD GLGRPLGPTP SQSRFQVDL VSENAGRAAA AAAAAAAAAA AAGAGAGAKQ TPADGEASGE SEPAKGSEEA KGRFRVNFVD PAASSSAEDS L SDAAGVGV ...String:
GAMGSEPRPT APSSGAPGLA GVGETPSAAA LAAARVELPG TAVPSVPEDA APASRDGGGV RDEGPAAAGD GLGRPLGPTP SQSRFQVDL VSENAGRAAA AAAAAAAAAA AAGAGAGAKQ TPADGEASGE SEPAKGSEEA KGRFRVNFVD PAASSSAEDS L SDAAGVGV DGPNVSFQNG GDTVLSEGSS LHSGGGGSGH HQHYYYDTHT NTYYLRTFGH NTMDAVPRID HYRHTAAQLG EK LLRPSLA ELHDELEKEP FEDGFANGEE STPTRDAVVT YTAESKGVVK FGWINGVLVR CMLNIWGVML FIRLSWIVGQ AGI GLSVLV IMMATVVTTI TGLSTSAIAT NGFVRGGGAY YLISRSLGPE FGGAIGLIFA FANAVAVAMY VVGFAETVVE LLKE HSILM IDEINDIRII GAITVVILLG ISVAGMEWEA KAQIVLLVIL LLAIGDFVIG TFIPLESKKP KGFFGYKSEI FNENF GPDF REEETFFSVF EIFFPAATGI LAGANISGDL ADPQSALPKG TLLAILITTL VYVGIAVSVG SCVVRDATGN VNDTIV TEL TNCTSAACKL NFDFSSCESS PCSYGLMNNF QVMSMVSGFT PLISAGIFSA TLSSALASLV SAPKIFQALC KDNIYPA FQ MFAKGYGKNN EPLRGYILTF LIALGFILIA ELNVIAPIIS NFFLASYALI NFSVFHASLA KSPGWRPAFK YYNMWISL L GAILCCIVMF VINWWAALLT YVIVLGLYIY VTYKKPDVNW GSSTQALTYL NALQHSIRLS GVEDHVKNFR PQCLVMTGA PNSRPALLHL VHDFTKNVGL MICGHVHMGP RRQAMKEMSI DQAKYQRWLI KNKMKAFYAP VHADDLREGA QYLMQAAGLG RMKPNTLVL GFKKDWLQAD MRDVDMYINL FHDAFDIQYG VVVIRLKEGL DISHLQGQEE LLSSQEKSPG TKDVVVSVEY S KKSDLDTS KPLSEKPITH KVEEEDGKTA TQPLLKKESK GPIVPLNVAD QKLLEASTQF QKKQGKNTID VWWLFDDGGL TL LIPYLLT TKKKWKDCKI RVFIGGKINR IDHDRRAMAT LLSKFRIDFS DIMVLGDINT KPKKENIIAF EEIIEPYRLH EDD KEQDIA DKMKEDEPWR ITDNELELYK TKTYRQIRLN ELLKEHSSTA NIIVMSLPVA RKGAVSSALY MAWLEALSKD LPPI LLVRG NHQSVLTFYS

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Macromolecule #2: POTASSIUM ION

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

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Macromolecule #3: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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Macromolecule #4: 3-(butylamino)-4-phenoxy-5-sulfamoylbenzoic acid

MacromoleculeName: 3-(butylamino)-4-phenoxy-5-sulfamoylbenzoic acid / type: ligand / ID: 4 / Number of copies: 2 / Formula: 82U
Molecular weightTheoretical: 364.416 Da
Chemical component information

ChemComp-82U:
3-(butylamino)-4-phenoxy-5-sulfamoylbenzoic acid / medication*YM / Bumetanide

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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: 7.4
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: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 1.175 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Number images used: 1033074

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-7s1y:
Cryo-EM structure of human NKCC1 K289NA492E bound with bumetanide

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