While the logo indicates which amino acids are acceptable in each position, it does not indicate which amino acids are unobserved. These are shown in the frequency matrix, and an example is also shown in Figure 2 for caspase-3. In this example Asp occurs in the P1 position in all 413 substrates, Asp occurs in P4 in almost half the substrates, while Glu occurs in P3 in 27% of substrates. Note that in this frequency matrix every amino acid occurs in positions P4-P2 and P1 0 -P4 0 , but tryptophan is observed only once in P4, P2, P1 0 and P4 0 . This gives an indication of the minimum number of substrate cleavages that has to be collected for a peptidase before definite conclusions about specificity in all binding pockets can be drawn.