Motivation: The analysis of sequence conservation patterns has been widely utilized to identify functionally important (catalytic and ligand-binding) protein residues for over a half-century. Despite decades of development, on average state-of-the-art non-template-based functional residue prediction methods must predict ∼25% of a protein's total residues to correctly identify half of the protein's functional site residues. The overwhelming proportion of false positives results in reported 'F-Scores' of ∼0.3. We investigated the limits of current approaches, focusing on the so-far neglected impact of the specific choice of homologs included in multiple sequence alignments (MSAs). Results: The limits of conservation-based functional residue prediction were explored by surveying the binding sites of 1023 proteins. A straightforward conservation analysis of MSAs composed of randomly selected homologs sampled from a PSI-BLAST search achieves average F-Scores of ∼0.3, a performance matching that reported by state-of-the-art methods, which often consider additional features for the prediction in a machine learning setting. Interestingly, we found that a simple combinatorial MSA sampling algorithm will in almost every case produce an MSA with an optimal set of homologs whose conservation analysis reaches average F-Scores of ∼0.6, doubling state-of-the-art performance. We also show that this is nearly at the theoretical limit of possible performance given the agreement between different binding site definitions. Additionally, we showcase the progress in this direction made by Selection of Alignment by Maximal Mutual Information (SAMMI), an information-theory-based approach to identifying biologically informative MSAs. This work highlights the importance and the unused potential of optimally composed MSAs for conservation analysis. Supplementary information: Supplementary data are available at Bioinformatics online.

Motivation: Multiple sequence alignments (MSAs) can provide essential input to many bioinformatics applications, including protein structure prediction and functional annotation. However, the optimal selection of sequences to obtain biologically informative MSAs for such purposes is poorly explored, and has traditionally been performed manually. Results: We present Selection of Alignment by Maximal Mutual Information (SAMMI), an automated, sequence-based approach to objectively select an optimal MSA from a large set of alternatives sampled from a general sequence database search. The hypothesis of this approach is that the mutual information among MSA columns will be maximal for those MSAs that contain the most diverse set possible of the most structurally and functionally homogeneous protein sequences. SAMMI was tested to select MSAs for functional site residue prediction by analysis of conservation patterns on a set of 435 proteins obtained from protein-ligand (peptides, nucleic acids and small substrates) and protein-protein interaction databases. Availability and implementation: A freely accessible program, including source code, implementing SAMMI is available at https://github.com/nelsongil92/SAMMI.git. Contact: [email protected]. Supplementary information: Supplementary data are available at Bioinformatics online.