|Entry||Database: PDB / ID: 6qp6|
|Title||Cryo-EM structure of calcium-bound mTMEM16F lipid scramblase in digitonin|
|Components||Anoctamin-6Calcium-dependent chloride channel|
|Keywords||MEMBRANE PROTEIN / lipid scrambles / TMEM16|
|Function / homology||Anoctamin, dimerisation domain / Dimerisation domain of Ca+-activated chloride-channel, anoctamin / Anoctamin-6 / Calcium-activated chloride channel / Neutrophil degranulation / Stimuli-sensing channels / Anoctamin / calcium activated phosphatidylserine scrambling / calcium activated phosphatidylcholine scrambling / purinergic nucleotide receptor signaling pathway ...Anoctamin, dimerisation domain / Dimerisation domain of Ca+-activated chloride-channel, anoctamin / Anoctamin-6 / Calcium-activated chloride channel / Neutrophil degranulation / Stimuli-sensing channels / Anoctamin / calcium activated phosphatidylserine scrambling / calcium activated phosphatidylcholine scrambling / purinergic nucleotide receptor signaling pathway / calcium activated galactosylceramide scrambling / phosphatidylserine exposure on blood platelet / calcium activated phospholipid scrambling / positive regulation of potassium ion export across plasma membrane / phospholipid scramblase activity / plasma membrane phospholipid scrambling / intracellular calcium activated chloride channel activity / negative regulation of cell volume / positive regulation of ion transmembrane transport / bone mineralization involved in bone maturation / pore complex assembly / activation of blood coagulation via clotting cascade / calcium activated cation channel activity / voltage-gated chloride channel activity / bleb assembly / chloride transport / sodium ion transmembrane transport / cation transport / positive regulation of endothelial cell apoptotic process / voltage-gated ion channel activity / chloride channel complex / chloride transmembrane transport / positive regulation of bone mineralization / dendritic cell chemotaxis / positive regulation of phagocytosis, engulfment / positive regulation of monocyte chemotaxis / calcium ion transmembrane transport / blood coagulation / positive regulation of apoptotic process / protein homodimerization activity / plasma membrane / cytosol / Anoctamin-6|
Function and homology information
|Specimen source||Mus musculus (house mouse)|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.2 Å resolution|
|Authors||Alvadia, C. / Lim, N.K. / Clerico Mosina, V. / Oostergetel, G.T. / Dutzler, R. / Paulino, C.|
|Funding support||Switzerland , Netherlands , 2 items |
|Citation||Journal: Elife / Year: 2019|
Title: Cryo-EM structures and functional characterization of the murine lipid scramblase TMEM16F.
Authors: Carolina Alvadia / Novandy K Lim / Vanessa Clerico Mosina / Gert T Oostergetel / Raimund Dutzler / Cristina Paulino
Abstract: The lipid scramblase TMEM16F initiates blood coagulation by catalyzing the exposure of phosphatidylserine in platelets. The protein is part of a family of membrane proteins, which encompasses ...The lipid scramblase TMEM16F initiates blood coagulation by catalyzing the exposure of phosphatidylserine in platelets. The protein is part of a family of membrane proteins, which encompasses calcium-activated channels for ions and lipids. Here, we reveal features of murine TMEM16F (mTMEM16F) that underlie its function as a lipid scramblase and an ion channel. The cryo-EM data of mTMEM16F in absence and presence of Ca define the ligand-free closed conformation of the protein and the structure of a Ca-bound intermediate. Both conformations resemble their counterparts of the scrambling-incompetent anion channel mTMEM16A, yet with distinct differences in the region of ion and lipid permeation. In conjunction with functional data, we demonstrate the relationship between ion conduction and lipid scrambling. Although activated by a common mechanism, both functions appear to be mediated by alternate protein conformations that are at equilibrium in the ligand-bound state.
SummaryFull reportAbout validation report
|Date||Deposition: Feb 13, 2019 / Release: Mar 6, 2019|
|Structure viewer||Molecule: |
Downloads & links
Mass: 106367.727 Da / Num. of mol.: 2 / Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ano6, Tmem16f / Cell line (production host): HEK293T cells / Production host: Homo sapiens (human) / References: UniProt: Q6P9J9
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / Reconstruction method: single particle reconstruction|
|Component||Name: mTMEM16F / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT|
|Molecular weight||Value: 0.212 MDa / Experimental value: NO|
|Source (natural)||Organism: Mus musculus (house mouse)|
|Source (recombinant)||Cell: HEK293T cells / Organism: Homo sapiens (human)|
|Buffer solution||Details: 0.1% digitonin, 150 mM NaCl, 20 mM HEPES, pH 7.5 and 2 mM EGTA. 1 mM CaCl2 were added before freezing.|
|Specimen||Conc.: 3.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Details: at 5 mA / Grid material: GOLD / Grid mesh size: 300 / Grid type: Quantifoil R1.2/1.3|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 288 kelvins|
-Electron microscopy imaging
Model: Talos Arctica / Image courtesy: FEI Company
|Microscopy||Microscope model: FEI TALOS ARCTICA|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 49407 / Calibrated magnification: 49407 / Nominal defocus max: 3000 nm / Nominal defocus min: 300 nm / Calibrated defocus min: 300 nm / Calibrated defocus max: 3000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 microns / Alignment procedure: COMA FREE|
|Specimen holder||Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 105 kelvins / Temperature (min): 90 kelvins|
|Image recording||Average exposure time: 9 sec. / Electron dose: 52 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 7 / Number of real images: 5633|
|EM imaging optics||Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV|
|Image scans||Width: 3838 / Height: 3710 / Movie frames/image: 60 / Used frames/image: 1-60|
|Software||Name: PHENIX / Version: 1.14_3260: / Classification: refinement|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|Particle selection||Number of particles selected: 1348247|
|Symmetry||Point symmetry: C2|
|3D reconstruction||Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 219302 / Algorithm: BACK PROJECTION / Number of class averages: 9 / Symmetry type: POINT|
|Atomic model building||Ref space: REAL|
|Atomic model building||PDB-ID: 5YOB|
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