(A–B) Masked cryo EM density maps of afTMEM16 in the presence of 0.5 mM Ca2+ (A) or in Ca2+-free (B) conditions. For clarity, one monomer is shown in gray while the other is colored in red (A, 0.5 …
(A–F) Top:+0.5 mM Ca2+. Middle: 0 Ca2+. Bottom:+0.5 mM Ca2+ and + 5 mole% C24:0. (A) Size exclusion profile of afTMEM16 in MSP1E3 nanodiscs. (B) SDS gel of the indicated size exclusion peaks using …
(A) Unmasked final C1 map highlighting the non-centered positioning of the protein within the nanodisc in the presence (top) and absence (bottom) of Ca2+. Dashed red box is drawn around the nanodisc …
(A–C) Data processing flow chart for +Ca2+ (A), Ca2+-free (B), and +C24:0 Ceramide +Ca2+ (C). Particles picked from manually inspected micrographs were sorted with 2D and 3D classification in C1 …
(A–I) The cryo-EM density (gray mesh) is overlaid with the respective atomic models (sticks), Ca2+-bound afTMEM16 is in maroon (top panels), Ca2+-free is in cyan (middle panels) and Ca2+-bound …
Helices are shown as cylinders and the domain-swapped α8 from the other monomer is shown in red. Ca2+ ions are shown as blue spheres.
(A–B) Structural alignment of the atomic models of Ca2+-bound afTMEM16 in nanodiscs (purple) and nhTMEM16 in detergent (limon, PDBID 4WIS, Cα RMSD 1.92 Å) of hTMEM16K in detergent (light blue, PDBID …
Structural alignment of afTMEM16 in the presence of 0.5 mM Ca2+ (maroon) and absence of Ca2+ (cyan) (blue sphere). Left: conformational changes in the cytosolic domain, Right: conformational changes …
(A) Structural alignment of the TM10-α7 dimer interface of afTMEM16 in Ca2+-bound (maroon) and Ca2+-free (cyan) conformations.
(A) Structural alignment of the lipid pathway with afTMEM16 in the presence (maroon) or absence (cyan) of 0.5 mM Ca2+. The color scheme is the same throughout the figure. Arrows indicate direction …
(A–B) The position of recently identified residues important for lipid scrambling by nhTMEM16 (red sticks) (Jiang et al., 2017; Lee et al., 2018) are mapped onto the afTMEM16 Ca2+-bound (A) and Ca2+-…
Structural alignment of TMEM16A in the presence of 0.5 mM Ca2+ (PDBID: 5OYB, red) and absence of Ca2+ (PDBID: 5OYG, blue). Left: conformational changes in ion permeation pathway. Right: view of …
(A) Close up view of the Ca2+-binding site, with key coordinating residues shown as sticks. The density corresponding to the Ca2+ ions (blue spheres) from the experimental map is shown in black and …
(A and C) The unmasked maps (gray) of the afTMEM16/nanodisc complex in the presence (A) and absence of Ca2+ (C) are shown low pass filtered to 10 Å at σ = 0.4 with front (left panels) and back …
(A–C) The unmasked maps of the afTMEM16/nanodisc complex in the presence of 0.5 mM Ca2+ (A), in Ca2+-free conditions (B) and with 0.5 mM Ca2+ and 5 mol% C24:0 Ceramide (C) are shown low-pass …
(A–C) The unmasked maps of afTMEM16/nanodisc complex in the presence of 0.5 mM Ca2+ determined from three datasets are shown low-pass filtered to 10 Å and at σ = 3.5. The high-resolution afTMEM16 …
(A–C) The unmasked maps of the afTMEM16/nanodisc complex in the presence of 0.5 mM Ca2+ determined from dataset A (see Supplementary file 1) are shown low-pass filtered to 10 Å and at σ = 3.5. The …
(A–B) Close-up view of representative 2D classes (box size 275 Å) of Ca2+-bound (0.5 mM) (top) and Ca2+-free (bottom) nanodisc-reconstituted afTMEM16 (taken from Figure 1 —figure supplement 1D). …
(A) Schematic of the in vitro scramblase assay. Liposomes are reconstituted with NBD-labeled phospholipids (green) that distribute equally in the two leaflets. Addition of extraliposomal sodium …
(A–D) Representative time courses of dithionite-induced fluorescence decay in protein-free liposomes (green) or in afTMEM16 proteoliposomes with (red) or without (black) Ca2+. Cyan dashed lines …
(A–D) Representative time courses of dithionite-induced fluorescence decay in control liposomes formed from a 3 POPE: 1 POPG mixtures with C18:0 Ceramide (A), C22:0 Ceramide (B), C24:0 Ceramide (C) …
(A) Structural alignment of Ca2+-bound afTMEM16 with (light blue) and without C24:0 ceramide (maroon) with a Cα r.m.s.d. <1 Å. (B) The unmasked maps (gray) of the afTMEM16/nanodisc complex in the …
Atomic model of afTMEM16 in in the presence of 0.5 mM Ca2+ and 5 mole% C24:0 Ceramide with location of lipid acyl chains within the side dimer cavity. Insets show acyl chains colored by ligand ID …
(A–D) Ca2+-dependent gating scheme for TMEM16 scramblases. The α helices lining the lipid pathway and Ca2+-binding site (TM3-8) are shown as cylinders. The two gating elements (TM3-4 and TM6) are …
(A–B) Alignment of Ca2+-bound afTMEM16 (maroon) and Ca2+-bound TMEM16A (red, PDBID: 5OYB). A close-up view of the pathway is shown in (B) to highlight the closure of the permeation pathway in the …
(A–C) Structural alignment of the dimer cavities of nhTMEM16 (purple) (A), hTMEM16K (green) (B), or mTMEM16A (red) (C) to afTMEM16 (maroon). The transmembrane region of afTMEM16 is shown as ribbon. …
(A–B) The unmasked maps of the afTMEM16/nanodisc complex in the presence of 0.5 mM Ca2+ (A) and of the apo SthK potassium channel/nanodisc complex (Rheinberger et al., 2018) are shown low-pass …
Morph between Ca2+-free and Ca2+-bound conformations of afTMEM16. For clarity one monomer is shown in gray. In the other, the cytosolic domain is in orange, the lipid permeation pathway (TM3-7) in …
Close-up view of the rearrangements undergone by the afTMEM16 lipid pathway (TM3-7) upon Ca2+ binding. Note that upon Ca2+ binding TM4 straightens and moves out of the pathway, TM3 slides downward …
Close-up view of the rearrangements undergone by the afTMEM16 ligand binding site upon Ca2+ binding. TM6 and TM7 are shown in green and TM8 is shown in blue. The residues directly involved in Ca2+ …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Biological sample (Saccharomyces cerevisiae) | FYG217 -URA | doi: 10.1038/nprot.2008.44 | cells for expression, URA3 deletion | |
Biological sample (Escherichia coli) | BL21(DE3) | Stratagene | Cells for MSP1E3 expression | |
Recombinant DNA reagent (synthetic gene) | MSP1E3 | Addgene https://www.addgene.org/20064/ | ||
Recombinant DNA reagent (synthetic gene) | pET 28a | Addgene https://www.addgene.org/20064/ | ||
Recombinant DNA reagent | pDDGFP2 (expression vector) | doi: 10.1038/nprot.2008.44 | - | |
Recombinant DNA reagent (Aspergillus fumigatus) | afTMEM16 | doi:10.1038/ncomms3367 | Gene ID: 3504033 | |
Software | Ana | M. Pusch, Istituto di Biofisica, Genova, Italy | http://users.ge.ibf.cnr.it/pusch/programs-mik.htm | |
Software | SigmaPlot | RRID:SCR_003210 | ||
Software | Leginon | doi: 10.1016/j.jsb.2005.03.010 | ||
Software | Relion | doi: 10.7554/eLife.18722 | ||
Software | MotionCorr2 | doi: 10.1038/nmeth.4193 | ||
Software | CTFFIND4 | doi: 10.1016/j.jsb.2015.08.008 | ||
Software | cryoSPARC | doi: 10.1038/nmeth.4169 | ||
Software | cisTEM | doi: 10.7554/eLife.35383 | ||
Software | Bsoft (Blocres) | doi: 10.1016/j.jsb.2006.06.006 | ||
Software | UCSF chimera | doi: 10.1002/jcc.20084 | ||
Software | pymol | Schrödinger |
Raw data of the representative fluorescence decay traces of afTMEM16-mediated lipid scrambling in liposomes formed from lipids with different chain length and saturation.
Raw data of the representative fluorescence decay traces of afTMEM16-mediated lipid scrambling in liposomes containing different Ceramide lipids.
Summary of cryo-EM datasets utilized in this work.
Detailed processing procedures are described in the methods. Note that +Ca2+dataset C was analyzed independently only up to 2D classification; after 2D classification it was combined with dataset B to generate dataset A, which yielded the final high-resolution +Ca2+ map.
Average values of the scrambling rate constants of afTMEM16 in short (16–18C) and long (22:1) chain lipids.
The following parameters were derived as described 6 by fitting the data to Eq. 1: f0 is the fraction of empty liposomes, α and β are the forward and reverse scrambling rate constants, γ is the reduction rate constant by dithionite, LiPF is the fraction of NBD-labeled lipids in the inner leaflet of a protein-free vesicle, n is the number of independent experiments. Data is reported as the mean ±SD. * denotes values that were constrained during fitting.
Statistics of cryo-EM data collection, 3D reconstruction and model refinement.
Average values of the scrambling rate constants of afTMEM16 in the presence of various ceramides.
The following parameters were derived by fitting the data to Eq. 1: f0 is the fraction of empty liposomes, α and β are the forward and backward scrambling rate constants, γ is the reduction rate constant by dithionite, LiPF is the fraction of NBD-labeled lipids in the inner leaflet of a protein-free vesicle, n is the number of independent experiments. Data is reported as the mean ±SD.