Aligned hydrophobic cluster analysis (HCA) plots of the catalytic domains of two GH45 proteins and domain 1 of an α-expansin protein, Arabidopsis EXPA15, with additional annotation based on the crystal structure of Humicola GH45. The GH45 sequences are from Humicola insolens (GenBank accession number P43316) and Trichoderma reesei (AAQ21385). HCA plots were constructed with DrawHCA . In these plots, the amino-acid sequence of each protein is written out in duplicate in a helical representation that puts together amino-acid residues that would be next to each other in an α helix. The six β sheets that form a barrel in the GH45 from Humicola (see Figure 5a) are indicated by boxes above the plot. Cytosine residues involved in intramolecular bridges and conserved in expansins and GH45 proteins are shown by blue dots connected by blue lines, also above the plot. Selected conserved motifs are highlighted in pink and the differences in their relative positions between proteins are indicated by black lines between the plots. The interpretation of HCA plots is summarized in . HCA uses the standard one-letter amino acid abbreviations except for four amino acids, as shown in the key. Hydrophobic residues are outlined. Clusters of hydrophobic residues are usually associated with regular secondary structures (α helices or β sheets). Zigzagging vertical lines of hydrophobic residues indicate alternating hydrophobic and non-hydrophobic residues, typical of exposed β sheets (for example, β2, β3, β5 and β6). Continuous hydrophobic clusters are more common in internal β sheets (for example, β4). Conservation of clusters and sequence motifs suggests that the core β-barrel structure with stabilizing cysteine bridges is conserved in the three proteins and that the differences are mostly in the size of the intervening loops. In Humicola GH45, the loops between β1 and β2 and between β5 and β6 have expanded considerably, while the other loops appear reduced in comparison with Trichoderma GH45. The latter appears more similar to expansin domain 1, which has an even more compact structure.