Computational protocol: Phylogenetic Analysis of Six-Domain Multi-Copper Blue Proteins

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Protocol publication

[…] Amino acid sequences were obtained from the GenPept protein sequences database (http://www.ncbi.nlm.nih.gov/protein/) using the BLASTP program . Cp (NP_036664.1) and MnxG (ZP_02951893.1) sequences were used as queries. Sequences with E-value < 0.001 were chosen as significantly similar. In order to exclude we one-, two- and three-domain MCBPs we selected only mononuclear and/or trinuclear copper binding sites containing sequences over 800 amino acids long. Multiple alignment of amino acid sequences was made using Seaview software using MUSCLE algorithm . Alignments of prokaryotic and eukaryotic MCBP were overlapped manually using BioEdit software on the basis of the conservative copper binding sites positions. The obtained alignments were used for phylogenetic analysis in MEGA4 using neighbor-joining method, phylogenetic distances were evaluated using Poisson correction model . Positions with alignment gaps were eliminated for pairwise alignments. Topology reliability was checked using bootstrap analysis on the basis of 1000 replicates. Protein sequences were designated in accordance with GenPept accession numbers. Organisms were classified in accordance with NCBI taxonomic hierarchy (http://www.ncbi.nlm.nih.gov/Taxonomy). […]

Pipeline specifications

Software tools BLASTP, SeaView, MUSCLE, BioEdit, MEGA
Applications Phylogenetics, Nucleotide sequence alignment
Chemicals Copper