MAST output containing the top 3 motifs identified by MEME.

<p>The figure shows sequences with motif E-values < 10⁻³⁹. Motif 1 (red boxes) maps to TMSs 7 and 8, where 4 of the 6 Ca²⁺-binding residues in nhTMEM16 are located. Motif 2 (cyan boxes) maps to TMSs 4 and 5 in nhTMEM16, which form part of the subunit cavity for phospholipid translocation. Motif 3 (green boxes) maps to TMS 1, but this TMS does not interact with Ca²⁺ or the substrate. Our results show that 94% (65/69) of the sequences in the superfamily map Motif 1 to the region that contains 4 of the 6 functional residues that bind Ca²⁺, and 98.5% (68/69) of the sequences map Motif 2 to TMSs 4 and 5.</p

Average topological features of the seven families within the Anoctamin Superfamily.

<p>Plots for all families were generated with the AveHAS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192851#pone.0192851.ref106" target="_blank">106</a>] program. Each plot is composed of two curves. Top dark red lines represent average hydropathy. Bottom gray dotted lines represent average similarity. Predicted TMSs are shown as vertical gray lines. Numbered bars above the hydropathy curves indicate the positions of peaks of hydrophobicity, usually predicted to be TMSs using the HMMTOP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192851#pone.0192851.ref096" target="_blank">96</a>] and WHAT [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192851#pone.0192851.ref095" target="_blank">95</a>] programs. This figure shows that there are 8 to 10 hydrophobicity peaks in all seven families, which likely correspond to 9 or 10 TMS, since, in this superfamily, some hydrophobicity peaks (such as peak 7 in <b>A</b>) are composed of 2 TMSs. The similarity curves indicate that the regions containing TMSs have the highest levels of conservation, and the corresponding multiple alignments shows that they have fewer gaps.</p

GSAT pairwise alignment of a homolog of the CSC-L1 family (XP_001010624) with a homolog of the CSC family (XP_014661822).

<p>The alignment shows the local region identified by Protocol2 that was used as evidence for homology between these two families. Family CSC-L1 has TC: 1.A.17.3 while family CSC has TC: 1.A.17.5. Notice that despite the low identity levels (22.7%), the TMSs align well, and a hydrophilic region between the second and third TMSs is shared (GSAT score 34.2 SD). TMSs were identified by running HMMTOP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192851#pone.0192851.ref096" target="_blank">96</a>] on the full protein sequences and then mapping the TMS coordinates in the alignment.</p

Predicted topologies and domain organizations of various members of the Anoctamin Superfamily.

<p>Open rectangular bars denote the positions of hydrophobic peaks, indicating putative TMSs. The locations of recognized Pfam domains are shown below thick gray lines representing the protein sequences.</p

Average protein sizes, numbers of predicted TMSs (based on average hydropathy plots) and source phyla for each of the seven major families in the Anoctamin Superfamily.

<p>Average protein sizes, numbers of predicted TMSs (based on average hydropathy plots) and source phyla for each of the seven major families in the Anoctamin Superfamily.</p

Phylogenetic tree of protein members of the Anoctamin Superfamily.

<p>The tree was generated with MrBayes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192851#pone.0192851.ref098" target="_blank">98</a>]. The multiple alignment used to build this tree was generated with MAFFT [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192851#pone.0192851.ref104" target="_blank">104</a>] and trimmed with trimAL [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192851#pone.0192851.ref105" target="_blank">105</a>] to ensure that each residue position in the alignment contained less than 15% gaps. The seven families are labeled as indicated in the text. The labels of the leaves correspond to the last 2 components of their TC identifier. Complete TC identifiers result from inserting “1.A.17.” to the left of each leaf label.</p

Top GSAT scores (expressed in standard deviations (SD)) between members of the seven families in the Anoctamin Superfamily^†.

<p>The inference of homology is based on the Superfamily Principle. See the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192851#sec019" target="_blank">Methods</a> section for procedural details. The table shows only the highest scores (columns 5–7) that allow the identification of homology transitivity paths A→B→C→D<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192851#t002fn001" target="_blank">^†</a> (columns 1–4) among all seven families. For each row, the cell corresponding to the comparison score in the transitivity path is shaded (lowest score; see columns 5–7). Notice how families in rows 1, 4, 5, 7 and 8 are related by the same protein; that is B = C, which indicates that the same protein has significant alignments with both Family 1 (A; column 1) and Family 2 (D; column 4).</p

Average hydropathy plot (dark red line) showing the basis for the topological predictions made for the <i>Nectria haematococca (Fusarium solani)</i> nhTMEM16 (anoctamin) protein (TC: 1.A.17.1.18), for which x-ray structures are available (PDB IDs 4WIS and 4WIT).

<p>Vertical tan bars show the positions of the predicted TMSs using the Loop Finder program (V. S. Reddy and M. H. Saier, unpublished). The green bar shows the position of the α-helix corresponding to TMS 6. This helix was not predicted to be a TMS by this program, HMMTOP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192851#pone.0192851.ref096" target="_blank">96</a>] or CCTOP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192851#pone.0192851.ref108" target="_blank">108</a>], although the x-ray structure confirmed that it is one. HMMTOP predicted TMSs 1 and 2 as a single TMS, although the structure confirms that the corresponding hydrophobicity peak is composed of two TMSs. The two purple bars, representing the position of transmembrane helices 7 and 8 in the x-ray structure, were predicted by these programs and AveHAS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192851#pone.0192851.ref106" target="_blank">106</a>] to be a single TMS (also note the 7^th hydrophobicity peak in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192851#pone.0192851.g004" target="_blank">Fig 4A</a>). This explains the discrepancy in the predictions for different members of the Anoctamin Superfamily (between 8 and 10 TMSs). The locations, in the hydropathy curve, of the three pairs of functional residues that bind Ca²⁺ in TMSs 6, 7 and 8 are depicted with blue, black and green circles, respectively.</p