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Progressively larger multiple sequence alignments were built by constructing multiple sequence alignments of each subfamily, which were manually polished and added together stepwise.

For each individual protein in the complex, we constructed multiple sequence alignments by querying the UniProt sequence database (Wu et al., 2006) for homologous sequences.

This unique feature is essential for our " on-the-fly" pipeline, which needs to classify a query sequence uploaded online by a user temporarily without re-constructing multiple sequence alignments.

The sequence information was used to construct multiple sequence alignment using TARGeT-based multiple sequence aligner.

First, we constructed multiple sequence alignments (MSAs) for each of the 19,778 human protein sequences defined by the Human Proteome Initiative (HPI) and their potential vertebrate homologs.

The degree of conservation and the phylogenetic relationship between Cif homologs were analysed by constructing a multiple sequence alignment and a phylogenetic tree using the Neighbour-Joining method (Fig. 1).

This can be done either by constructing a multiple sequence alignment from the best matching hits with subsequent phylogenetic reconstruction, as implemented in Phylogena [ 58], or it can be done directly based on the BLAST results.

HMMs for each cluster were constructed by iteratively constructing multiple alignments with Kalign [ 34] and removing columns represented in less than 10% of the sequences, or in at least two sequences if the cluster contains less than 10 sequences.

CLUSTALX was used for constructing multiple alignments of the sequences.

We represent variability in sequence conservation by an entropy profile – a vector constructed from multiple sequence alignment where the value at the ith position in the vector equals the entropy of the corresponding column in the alignment.

We then constructed a multiple sequence alignment (MSA) of the sequences using automated alignment tools followed by manual correction (see Methods).