*2.3. Multiple Sequence Alignments and Three-Dimensional Structure of Apple A20/AN1 Domains*

Multiple alignments demonstrated that the A20/AN1 domains are conserved among the *MdSAP* proteins (Figure 2). We then produced sequence logos that further showed that these domains were highly conserved at each residue position (Figure 3A,C; Supplementary Sequences A2 and A3). Afterward, the SWISS-MODEL web server was used for modeling and analysis of homology among protein structures. For this, we built the A20 domain and AN1 domain homology models and evaluated them using the homologous templates 2KZY.pdb and 1WFP, respectively (Figure 3B,D). The 3D models indicated that, respectively, the A20 domain and the AN1 domain in the *MdSAP7* structure most closely matched the A20 domain of ubiquitin receptor ZNF216 and the zf-AN1 domain of the *Arabidopsis* F5O11.17 protein (PDB ID: 2KZY.1.A, 38% sequence identity for residues 18–50; 1WFP.1.A, 47% sequence identity for residues 96–148).

**Figure 2.** Multiple alignments of A20/AN1 domain and C2H2 amino acids in apple SAPs, using the DNAMAN program. Conserved domains are boxed, and identical amino acids are shown against a dark blue background (Similarity: dark blue = 100%; pink > 75%; cyan > 50%).

**Figure 3.** (**A**) Sequence logos for A20 domain in 26 *MdSAP* proteins, generated via WebLogo; (**B**) Three-dimensional tertiary structural model of A20 domain (PDB ID: 2KZY.1.A); (**C**) Sequence logos of AN1 domain in 28 *MdSAP* proteins; and (**D**) Three-dimensional tertiary structural model of AN1 domain (PDB ID: 1WFP.1.A). Within each stack, symbol height indicates the relative frequency of each amino acid at that position. Logos for the A20 domain and AN1 domain were obtained through multiple alignments of 26 and 28 *MdSAP* protein sequences, respectively. At the top of the corresponding amino acid sequences, arabic numbers (1–5) indicate β-sheets in A20 and AN1 domains. In (**B**,**D**), α-helices are red, β-sheets (arabic numbers 1–5) are yellow, and strands are blue/gray. Three-dimensional representations were generated with RasTop software.
