|Entry||Database: PDB / ID: 6qm5|
|Title||Cryo-EM structure of calcium-bound nhTMEM16 lipid scramblase in DDM|
|Keywords||MEMBRANE PROTEIN / lipid scrambles / TMEM16|
|Function / homology||Anoctamin / Calcium-activated chloride channel / integral component of membrane / identical protein binding / metal ion binding / Uncharacterized protein|
Function and homology information
|Biological species||Nectria haematococca (fungus)|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å|
|Authors||Kalienkova, V. / Clerico Mosina, V. / Bryner, L. / Oostergetel, G.T. / Dutzler, R. / Paulino, C.|
|Funding support|| Switzerland, Netherlands, 2items |
|Citation||Journal: Elife / Year: 2019|
Title: Stepwise activation mechanism of the scramblase nhTMEM16 revealed by cryo-EM.
Authors: Valeria Kalienkova / Vanessa Clerico Mosina / Laura Bryner / Gert T Oostergetel / Raimund Dutzler / Cristina Paulino /
Abstract: Scramblases catalyze the movement of lipids between both leaflets of a bilayer. Whereas the X-ray structure of the protein nhTMEM16 has previously revealed the architecture of a Ca-dependent lipid ...Scramblases catalyze the movement of lipids between both leaflets of a bilayer. Whereas the X-ray structure of the protein nhTMEM16 has previously revealed the architecture of a Ca-dependent lipid scramblase, its regulation mechanism has remained elusive. Here, we have used cryo-electron microscopy and functional assays to address this question. Ca-bound and Ca-free conformations of nhTMEM16 in detergent and lipid nanodiscs illustrate the interactions with its environment and they reveal the conformational changes underlying its activation. In this process, Ca binding induces a stepwise transition of the catalytic subunit cavity, converting a closed cavity that is shielded from the membrane in the absence of ligand, into a polar furrow that becomes accessible to lipid headgroups in the Ca-bound state. Additionally, our structures demonstrate how nhTMEM16 distorts the membrane at both entrances of the subunit cavity, thereby decreasing the energy barrier for lipid movement.
SummaryFull reportAbout validation report
|Structure viewer||Molecule: |
Downloads & links
A: Predicted protein
B: Predicted protein
Mass: 83200.008 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nectria haematococca (fungus) / Gene: NECHADRAFT_66456 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: C7Z7K1
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction|
|Component||Name: nhTMEM16 / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT|
|Molecular weight||Value: 0.166 MDa / Experimental value: NO|
|Source (natural)||Organism: Nectria haematococca mpVI 77-13-4 (fungus)|
|Source (recombinant)||Organism: Saccharomyces cerevisiae (baker's yeast)|
|Buffer solution||pH: 7.6 |
Details: 5 mM Hepes 7.6, 150 mM NaCl, 0.03% DDM, 2 mM EGTA. 2.3 mM CaCl2 were added 30 minutes 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 divisions/in. / Grid type: Quantifoil R1.2/1.3|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 288 K|
-Electron microscopy imaging
Model: Talos Arctica / Image courtesy: FEI Company
|Microscopy||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 X / Calibrated magnification: 49407 X / 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 µm / Alignment procedure: COMA FREE|
|Specimen holder||Cryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 105 K / Temperature (min): 90 K|
|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) / Num. of grids imaged: 9 / Num. of real images: 2521|
|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||Num. of particles selected: 251693|
|Symmetry||Point symmetry: C2 (2 fold cyclic)|
|3D reconstruction||Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 120086 / Algorithm: BACK PROJECTION / Symmetry type: POINT|
|Atomic model building||Space: REAL|
|Atomic model building||PDB-ID: 4WIS|
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