Computational protocol: Phylogeny of Rieske/cytb Complexes with a Special Focus on the Haloarchaeal Enzymes

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Protocol publication

[…] Protein sequences were collected by BLAST searches on completely sequenced genomes on the NCBI server. All available archaeal genomes were included in the analysis. For Bacteria, 5–10 species per phylum were selected, if available. Sequence alignments were done in several steps: A first alignment was done by Clustal (), and in a second step, the alignment was refined, taking into account the structural features of the protein, that were either taken directly from the 3D structure, when available, or in most cases predicted with the protein sequence analysis and modeling software package PSAAM (, last accessed July 26, 2012). With the help of Seaview (), the alignment was corrected to assure that corresponding structural elements (such as α-helices or β-sheets) were properly aligned. Each of these structural elements and the connecting sequence stretches was then submitted individually to Clustal. This procedure was crucial for the Rieske alignment as explained in detail in a previous study () and was also used to improve the alignment of the cytochrome b sequences. N- or C-terminal extensions that occur in individual species were cut from this alignment before submitting it to tree-building procedures. In the case of cytochrome b, either the entire protein sequence or the first four transmembranous helices and their connecting sequence stretches, corresponding to cytochrome b6 in Cyanobacteria and chloroplasts, were used. Both sets of sequences gave similar results, but the less conserved C-terminal part of the protein induced a higher uncertainty reflected by lower bootstrap values. The “four-helix” alignment was used to calculate the trees of cytochrome b, shown in , , and . Different algorithms such as maximum likelihood, minimum evolution, or neighbor joining were tested to calculate the trees with the program MEGA (K. Tamura, D. Peterson, N. Peterson, M. Nei, and S. Kumar). The trees obtained with these different algorithms and with the neighbor joining algorithm () from Clustal, corrected for multiple substitutions, were essentially identical with respect to the branching order of the different phyla. Clustal was then used to calculate the trees depicted in this article. […]

Pipeline specifications

Software tools SeaView, MEGA
Application Protein sequence analysis
Chemicals Quinones