General methodsTo build a database among different domains we first needed an input database of protein structures classified into folds. We used the SCOPe database, releases 2.03 and 2.06. The SCOPe database is a database of domains of known structure according to structural and evolutionary relationships. It classifies protein domains hierarchically into levels, the two lowest (family and superfamily) capturing homologous relationships, while the upper levels, folds and classes, group superfamilies according to their topological connections. Let's specify the process we did for SCOPe 2.06. We took the SCOP95 subset, where sequences are filtered out to keep a maximum of 95% identity between any two domains. This input database contains 28010 domains. For each domain sequence we created a sequence alignment either using PSIblast. The multiple alignments served as base to build a profile HMM for each domain. We then performed an all-against-all pairwise HMM profile comparisons of the full domains in the sequence space alone, using HHsearch. At this stage, we had pairs of domains that share a protein segment with a certain homology. Subsequently, we measured the structural similarity of each pair using TM-align. We refer to these protein segments as fragments , i.e, significantly sized protein segments with remote homology and similar structure. This pipeline led to a final set of over 8 million pairs (termed hits herein) that we stored into our Fuzzle database, standing for Fold Puzzle.
A look inside the databaseInternally, each hit is comprised by the two domain SCOP identifiers (query and subject), their SCOPe families, the HHsearch probability of the homologous protein fragment, sequence positions of the fragment, and the TM-score and RMSD of the structural alignment for the sequence fragment (see figure above). stored into this Fuzzle database, standing for Fold Puzzle. There are over 1.8 million hits where the two domains belong to different folds. We observed a bimodal distribution when plotting TM-score vs. HHsearch probabilities for pairs with domains belonging to different folds (high probability, high TM-score):
The set of hits in the upper left of the figure, i.e hits with high-sequence and structural similarity, reveals that that there are unrecognized homologous relationships. We therefore explored further this set of hits, those with probabilities over 70% and TM-score > 0.3. When representing this set of hits according to their length and probability (b), we observe most of the hits present short lengths (0 - 40 amino acids) and low probabilities. There are however, a non-negligible proportion of hits at larger lengths (50 – 70 amino acids) and some small areas with fragments over 100 amino acids.