Computational protocol: Restriction endonuclease BpuJI specific for the 5′-CCCGT sequence is related to the archaeal Holliday junction resolvase family

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[…] Fold recognition (an attempt to match sequence with known structures) was performed using HHpred server (http://protevo.eb.tuebingen.mpg.de/hhpred) (), which is based on a comparison of profile Hidden Markov Models (HMMs) (). To ensure consistency of the results in addition to the BpuJI CTD several other homologous sequences were analysed as well. Detection of sequence homologs of the BpuJI CTD was performed with saturated BLAST (), which is based on multiple intermediate (transitive) sequence searches. Each search consisted of PSI-BLAST () run of up to eight iterations using low complexity filtering and a conservative E-value cutoff (10−4) for sequence inclusion into the profile. Multiple sequence alignment was constructed using combination of PCMA () and ProbCons () followed by some manual adjustments to improve the hydrophobic packing of the core structure of the PD-(D/E)XK fold. The identification of additional conserved domains in the BpuJI CTD homologs was done using reverse-position-specific BLAST (RPS-BLAST) searches against the Conserved Domain Database (CDD) (). For the phylogenetic distribution analysis of BpuJI CTD-like domain in eukaryotes, additional BLAST searches were performed against translated NCBI databases of complete and incomplete genomes as well as EST databases. […]

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