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- PDB-6ukn: Cryo-EM structure of the potassium-chloride cotransporter KCC4 in... -
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Basic information
Entry | Database: PDB / ID: 6ukn | |||||||||
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Title | Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs | |||||||||
![]() | Solute carrier family 12 member 7 | |||||||||
![]() | TRANSPORT PROTEIN / Potassium chloride cotransporter / SLC12 | |||||||||
Function / homology | ![]() Cation-coupled Chloride cotransporters / ammonium import across plasma membrane / potassium:chloride symporter activity / chloride ion homeostasis / ammonium channel activity / potassium ion homeostasis / cell volume homeostasis / plasma membrane => GO:0005886 / potassium ion import across plasma membrane / chloride transmembrane transport ...Cation-coupled Chloride cotransporters / ammonium import across plasma membrane / potassium:chloride symporter activity / chloride ion homeostasis / ammonium channel activity / potassium ion homeostasis / cell volume homeostasis / plasma membrane => GO:0005886 / potassium ion import across plasma membrane / chloride transmembrane transport / chemical synaptic transmission / synapse / protein kinase binding / protein-containing complex Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.65 Å | |||||||||
![]() | Reid, M.S. / Kern, D.M. / Brohawn, S.G. | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs. Authors: Michelle S Reid / David M Kern / Stephen Graf Brohawn / ![]() Abstract: Cation-chloride-cotransporters (CCCs) catalyze transport of Cl with K and/or Naacross cellular membranes. CCCs play roles in cellular volume regulation, neural development and function, audition, ...Cation-chloride-cotransporters (CCCs) catalyze transport of Cl with K and/or Naacross cellular membranes. CCCs play roles in cellular volume regulation, neural development and function, audition, regulation of blood pressure, and renal function. CCCs are targets of clinically important drugs including loop diuretics and their disruption has been implicated in pathophysiology including epilepsy, hearing loss, and the genetic disorders Andermann, Gitelman, and Bartter syndromes. Here we present the structure of a CCC, the K-Cl cotransporter (KCC) KCC4, in lipid nanodiscs determined by cryo-EM. The structure, captured in an inside-open conformation, reveals the architecture of KCCs including an extracellular domain poised to regulate transport activity through an outer gate. We identify binding sites for substrate K and Cl ions, demonstrate the importance of key coordinating residues for transporter activity, and provide a structural explanation for varied substrate specificity and ion transport ratio among CCCs. These results provide mechanistic insight into the function and regulation of a physiologically important transporter family. | |||||||||
History |
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 111.4 KB | Display | ![]() |
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PDB format | ![]() | 80.8 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Summary document | ![]() | 796.3 KB | Display | ![]() |
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Full document | ![]() | 798.3 KB | Display | |
Data in XML | ![]() | 21.6 KB | Display | |
Data in CIF | ![]() | 30.6 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 20807MC M: map data used to model this data C: citing same article ( |
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Similar structure data | |
EM raw data | ![]() Data size: 1.1 TB Data #1: Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs [micrographs - multiframe]) |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 120610.961 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() | ||||||
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#2: Polysaccharide | Source method: isolated from a genetically manipulated source #3: Chemical | ChemComp-K / | #4: Chemical | ChemComp-CL / | Has ligand of interest | Y | |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: KCC4 in lipid nanodiscs / Type: CELL / Entity ID: #1 / Source: RECOMBINANT | ||||||||||||||||||||
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Source (natural) | Organism: ![]() ![]() | ||||||||||||||||||||
Source (recombinant) | Organism: ![]() ![]() | ||||||||||||||||||||
Buffer solution | pH: 8 | ||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||
Specimen support | Grid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3 | ||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 3uL, 1 blot force, 4s blot time, 1s wait time |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 36000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN |
Image recording | Average exposure time: 0.11 sec. / Electron dose: 0.9333 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
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Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1826000 | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.65 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 110143 / Symmetry type: POINT |