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- PDB-6d3s: Thermostabilized phosphorylated chicken CFTR -

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Basic information

Entry
Database: PDB / ID: 6d3s
TitleThermostabilized phosphorylated chicken CFTR
ComponentsCystic fibrosis transmembrane conductance regulator
KeywordsMEMBRANE 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 speciesGallus gallus (chicken)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.6 Å
AuthorsFay, J.F. / Riordan, J.R. / Chen, Z.J.
CitationJournal: 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.
Validation Report
SummaryFull reportAbout validation report
History
DepositionApr 16, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 17, 2018Provider: repository / Type: Initial release
Revision 1.1Nov 7, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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Assembly

Deposited unit
A: Cystic fibrosis transmembrane conductance regulator
hetero molecules


Theoretical massNumber of molelcules
Total (without water)163,6523
Polymers162,6371
Non-polymers1,0142
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Cystic fibrosis transmembrane conductance regulator / / ATP-binding cassette sub-family C member 7 / Channel conductance-controlling ATPase / cAMP- ...ATP-binding cassette sub-family C member 7 / Channel conductance-controlling ATPase / cAMP-dependent chloride channel


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: 3.6.3.49
#2: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: 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 solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
10.15 MSodium chlorideNaclSodium chloride1
20.04 MTris-HClTris1
30.005 MATP-Mg1
SpecimenConc.: 3.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 %

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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 FIELDBright-field microscopy / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron 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.

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameCategory
2SerialEMimage acquisition
4GctfCTF correction
7Rosettamodel fitting
9Rosettamodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 700000
3D reconstructionResolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 57826 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL / Target criteria: iFSC, elec_dens_fast

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