|Entry||Database: PDB / ID: 6d3s|
|Title||Thermostabilized phosphorylated chicken CFTR|
|Components||Cystic fibrosis transmembrane conductance regulator|
|Keywords||MEMBRANE PROTEIN / CFTR|
|Function / homology|
Function and homology information
positive regulation of cyclic nucleotide-gated ion channel activity / Sec61 translocon complex binding / positive regulation of voltage-gated chloride channel activity / intracellularly ATP-gated chloride channel activity / channel-conductance-controlling ATPase / transepithelial water transport / intracellular pH elevation / bicarbonate transmembrane transporter activity / multicellular organismal water homeostasis / chloride channel inhibitor activity ...positive regulation of cyclic nucleotide-gated ion channel activity / Sec61 translocon complex binding / positive regulation of voltage-gated chloride channel activity / intracellularly ATP-gated chloride channel activity / channel-conductance-controlling ATPase / transepithelial water transport / intracellular pH elevation / bicarbonate transmembrane transporter activity / multicellular organismal water homeostasis / chloride channel inhibitor activity / vesicle docking involved in exocytosis / membrane hyperpolarization / cholesterol transport / chloride channel complex / cellular response to forskolin / chloride transmembrane transport / positive regulation of exocytosis / sperm capacitation / cholesterol biosynthetic process / ATPase-coupled transmembrane transporter activity / positive regulation of insulin secretion involved in cellular response to glucose stimulus / response to endoplasmic reticulum stress / PDZ domain binding / cellular response to cAMP / recycling endosome / bicarbonate transport / transmembrane transport / chaperone binding / apical plasma membrane / early endosome / ATPase activity / endoplasmic reticulum membrane / cell surface / integral component of plasma membrane / enzyme binding / membrane / ATP binding / plasma membrane / cytosol
Cystic fibrosis TM conductance regulator (CFTR), regulator domain / ABC transporter transmembrane region / ABC transporter / ABC transporter type 1, transmembrane domain superfamily / P-loop containing nucleoside triphosphate hydrolase / CFTR regulator domain / ABC transporter, conserved site / ABC transporter type 1, transmembrane domain / Cystic fibrosis transmembrane conductance regulator / AAA+ ATPase domain / ABC transporter-like
Cystic fibrosis transmembrane conductance regulator / Cystic fibrosis transmembrane conductance regulator
|Biological species||Gallus gallus (chicken)|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.6 Å|
|Authors||Fay, J.F. / Riordan, J.R. / Chen, Z.J.|
|Citation||Journal: Biochemistry / Year: 2018|
Title: Cryo-EM Visualization of an Active High Open Probability CFTR Anion Channel.
Authors: Jonathan F Fay / Luba A Aleksandrov / Timothy J Jensen / Liying L Cui / Joseph N Kousouros / Lihua He / Andrei A Aleksandrov / Drew S Gingerich / John R Riordan / James Z Chen /
Abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, crucial to epithelial salt and water homeostasis, and defective due to mutations in its gene in patients with cystic ...The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, crucial to epithelial salt and water homeostasis, and defective due to mutations in its gene in patients with cystic fibrosis, is a unique member of the large family of ATP-binding cassette transport proteins. Regulation of CFTR channel activity is stringently controlled by phosphorylation and nucleotide binding. Structural changes that underlie transitions between active and inactive functional states are not yet fully understood. Indeed the first 3D structures of dephosphorylated, ATP-free, and phosphorylated ATP-bound states were only recently reported. Here we have determined the structure of inactive and active states of a thermally stabilized CFTR, the latter with a very high channel open probability, confirmed after reconstitution into proteoliposomes. These structures, obtained at nominal resolution of 4.3 and 6.6 Å, reveal a unique repositioning of the transmembrane helices and regulatory domain density that provide insights into the structural transition between active and inactive functional states of CFTR. Moreover, we observe an extracellular vestibule that may provide anion access to the pore due to the conformation of transmembrane helices 7 and 8 that differs from the previous orthologue CFTR structures. In conclusion, our work contributes detailed structural information on an active, open state of the CFTR anion channel.
SummaryFull reportAbout validation report
|Structure viewer||Molecule: |
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A: Cystic fibrosis transmembrane conductance regulator
|#1: Protein|| |
Mass: 162637.438 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Gene: CFTR / Cell line (production host): BHK / Production host: Cricetinae (hamsters)
References: UniProt: A0M8U4, UniProt: A0A1D5PBN0*PLUS, EC: 188.8.131.52
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: CELL / 3D reconstruction method: single particle reconstruction|
|Component||Name: CFTRCystic fibrosis transmembrane conductance regulator|
Type: CELL / Entity ID: #1 / Source: RECOMBINANT
|Source (natural)||Organism: Gallus gallus (chicken)|
|Source (recombinant)||Organism: Cricetinae (hamsters) / Cell: BHK / Plasmid: pNUT|
|Buffer solution||pH: 7.4|
|Specimen||Conc.: 3.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 %|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy / Alignment procedure: BASIC|
|Specimen holder||Cryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Image recording||Electron dose: 42 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 3410 |
Details: I had better girds but the vacuum crashed for the grids in the autoloader.
|Software||Name: PHENIX / Version: 1.13_2998: / Classification: refinement|
|CTF correction||Type: NONE|
|Particle selection||Num. of particles selected: 700000|
|3D reconstruction||Resolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 57826 / Num. of class averages: 1 / Symmetry type: POINT|
|Atomic model building||Protocol: OTHER / Space: REAL / Target criteria: iFSC, elec_dens_fast|
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