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- PDB-7s1x: Cryo-EM structure of human NKCC1 K289NA492EL671C bound with bumetanide -

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Basic information

Entry
Database: PDB / ID: 7s1x
TitleCryo-EM structure of human NKCC1 K289NA492EL671C bound with bumetanide
ComponentsSolute carrier family 12 member 2
KeywordsTRANSPORT PROTEIN / cation-chloride cotransporter / ion transport
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 / 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
3-(butylamino)-4-phenoxy-5-sulfamoylbenzoic acid / : / Solute carrier family 12 member 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsZhao, Y.X. / Cao, E.H.
Funding support1items
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, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 25, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Solute carrier family 12 member 2
A: Solute carrier family 12 member 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)264,6558
Polymers263,7772
Non-polymers8786
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Solute carrier family 12 member 2 / Basolateral Na-K-Cl symporter / Bumetanide-sensitive sodium-(potassium)-chloride cotransporter 2


Mass: 131888.641 Da / Num. of mol.: 2 / Mutation: K289N,A492E,L671C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC12A2, NKCC1
Production host: Mammalian expression vector BsrGI-MCS-pcDNA3.1 (others)
References: UniProt: P55011
#2: Chemical ChemComp-82U / 3-(butylamino)-4-phenoxy-5-sulfamoylbenzoic acid


Mass: 364.416 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C17H20N2O5S / Feature type: SUBJECT OF INVESTIGATION / Comment: medication*YM
#3: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: K
#4: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: human NKCC1 K289NA492EL671C bound with bumetanide / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.26334 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Mammalian expression vector BsrGI-MCS-pcDNA3.1 (others)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 1.175 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18_3845: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 504481
3D reconstructionResolution: 2.9 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Num. of particles: 504481 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00512954
ELECTRON MICROSCOPYf_angle_d0.78817664
ELECTRON MICROSCOPYf_dihedral_angle_d10.4521886
ELECTRON MICROSCOPYf_chiral_restr0.0512062
ELECTRON MICROSCOPYf_plane_restr0.0052216

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