Sequences of EF-hand motifs in Nox5, Duox, plant Nox, and NoxC. (A) EF-hand containing Nox/Duox family members are listed and demonstrate the widespread occurrence of these Noxes. Parentheses represent the presence of Nox5 ortholog in the T. nigroviridis and D. rerio genomes, which clearly belongs in the Nox5 subgroup (#45 and #47 in Figure 2), but appears to lack the N-terminal EF-hand-containing domain as discussed in the text. (B) Based on alignments shown in Additional files 2 and 8, the arrangement of EF-hand motifs within the calcium-binding domain of calcium-regulated Noxes are shown schematically. EF-I to EF-VI in squares indicate each canonical EF-hand motif. TM-I and TM-II indicate the 1st or 2nd TM segments. Asterisks represent atypical EF-hand motifs that differ from consensus sequences at positions 1, 3, or 12, normally the most conserved positions . HLH, refers to a non-canonical helix-loop-helix predicted structure. (C) A structural homology model of the EF-hand-containing domains of human Nox5 (upper panel) and human Duox1 (lower panel) using a comparative protein modeling method (SWISS-MODEL) and visualized with Deep View Swiss-PDB. The N-terminal region of Nox5 and human Duox1 was calculated using the structure of calcineurin B subunit isoform 1 as a fit template. The fit of the N-terminal region of Duox1 corresponding to the first EF-hand-like motif of Nox5 was not accurate enough to determine the molecular model. The arrow indicates the position of Duox1 corresponding to the 4th EF-hand motif of Nox5 and models as a HLH structure that lacks canonical calcium binding amino acid residues. Side chains of canonical EF-hand motifs are indicated. Conserved sequences among EF-hand regions in Noxes and Duoxes are aligned and compared in Additional file 2.