Computational protocol: Functional and evolutionary characterization of Ohr proteins in eukaryotes reveals many active homologs among pathogenic fungi

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Protocol publication

[…] The amino acid sequence from Xylella fastidiosa 9a5c strain was used as query for search against NCBI nr sequence database using delta-BLAST via NCBI website (June of 2016) and the profile Hidden Markov Model (HMM) iterative method implemented in Jackhmmer 1.9 web server . We conducted the search against NCBI nr sequence database using default options until convergence. The searches were restricted to the Eukarya Domain. Redundant entries and truncated sequences (less than 100 amino acids) were removed using CD-HIT software . [...] We identified members of the Ohr and OsmC subfamilies in Eukarya using sequence motifs previously described , . Additional motifs were detected using alignments of eukaryotic Ohr sequences with structurally solved Ohr (PDB: 1ZB8, from X. fastidiosa; 1USP, from Deinococcus radiodurans; 3LUS, from Vibrio cholerae; 1N2F, from Pseudomonas aeruginosa) or OsmC (1NYE, from Escherichia coli) proteins. We curated the alignments manually, guided by successive multiple alignments runs generated by MAFFT operating with default sets . The input sequences were collected by delta-blast and jackhmmer searches described in the previous section. [...] Maximum Likelihood (ML) inference of phylogenetic trees was based on the manually curated MAFFT alignment and the RAxML software and applied to all non-redundant sequences retrieved or only sequences from the Ohr subfamily. For inference, we used Whelan-Goldman (WAG) model of amino acid evolution with rate heterogeneity modeled by a GAMMA distribution and 1000 rapid bootstrap resampled estimates of log-likelihood (RELL bootstrap). The resulting phylogeny was prepared for visualization using Tree Editor from the MEGA 7 software . […]

Pipeline specifications

Software tools DELTA-BLAST, jackhmmer, CD-HIT, MAFFT, RAxML, tred, MEGA
Applications Phylogenetics, Amino acid sequence alignment
Organisms Musa acuminata, Bacteria
Chemicals Glutathione, Hydrogen Peroxide