Fig. 5.

Sequence alignments highlighting the amphipathic helix insertions of the NuoL subunits from 2M complexes and their close relatives. Cladograms represent the same tree topology as those in Fig. 4, with colored branches marking the 2M groups. The T. thermophilus sequence is included in the alignment as a sequence length metric of where the amphipathic helix begins and ends. a Clade 1 contains 26–28 amino acids insertions compared to many of the close relatives and T. thermophilus. b Clade 2 contains 27 amino acid insertions compared to their closest relatives (28 compared to T. thermophilus). c Clade 3 contains 26 amino acid insertions. Notably, in a and c the closest relatives of the 2M complexes that were used as outgroups in the phylogenetic analysis also have insertions in the amphipathic helix, despite not having a second distantly related NuoM in their complex I operons. MUSCLE alignments are shown here, Clustal Omega and MAFFT alignments are shown in Figures S32-S34