The most reliable protein sequence alignments may be obtained when all the proteins have had their tertiary structures experimentally determined. Comparison of three dimensional structures also allows much more distantly related proteins to be aligned accurately. Analysis of such alignments should therefore give the best substitution matrices. Accordingly, Risler et al.  derived substitution frequencies from 32 proteins structurally aligned in 11 groups. On similar lines, Overington et al.  aligned 7 families for which 3 or more proteins of known three dimensional structure were known and derived a series of substitution matrices. Overington et al. also subdivided the substitution data by the secondary structure and environment of each amino acid, however this led to rather sparse matrices due to the lack of examples. Bowie et al.  have also derived substitution tables specific for different amino acid environments and secondary structures.