Several MRP4-related PDB structures were found from the PSI-BLAST search with experimentally characterized PDB structures, although there is no solved PDB structure of MRP4 elucidated so far. YASARA Structure selected the templates, such as Bos taurus MRP1 (PDB ID: 5UJA) with 35.2% identity, Caenorhabditis elegans P-gp (PDB ID: 4F4C) with 19.1% identity, MmMRP1 (PDB ID: 4M1M) with 18.9% identity, and MmMRP1 (PDB ID: 4Q9H) with 18.3% identity. Each homology model, built with each template, was refined by unrestrained high-resolution energy minimization using the latest knowledge-based YASARA force field . As a final model, a hybrid homology model was assembled by combining the best scoring parts of the four models and then refined with energy minimization. Out of 1469 residues, 1425 residues were modeled, omitting 44 C-terminal residues, since YASARA Structure does not perform ab initio or threading modeling (Fig. 2).Fig. 2Structural characterization and conservation of CsMRP4. a A 3D homology model was built based on the solved structure templates. The α-helix and β-strand are depicted as ribbon diagrams, and coiled-coil is depicted as a line. TM α-helices are colored and numbered from the N-terminus (blue) to the C-terminus (red). The red rectangles indicate the entrance of inner cavity, and spheres correspond to residues coordinating ATP-binding sites. b TM α-helices, as viewed perpendicular to the horizontal plane marked with red arrowheads. c The degree of sequence conservation is colored using a color gradient from white (divergent) to red (conserved). Structural conservation corresponds to the radii of the backbone sausage representation, which is proportional to the root-mean-squared deviation at each position between structure alignments. PDB IDs of the identified homologs are as follows: 5W81_A, 5UAK_A, 5UJ9_A, 4C3Z_A, 2PZG_A, 3GD7_A, 4Q4J_B, 1R0Z_A, 2HYD_A, 4Q7M_B, 4Q4J_A, 5DGX_A, 5IDV_A, 4MYC_A, 3WMF_A, 5MKK_A, 1MV5_A, 4MRN_A, 3NH6_A, 2FFB_A, 5EUM_A, 4F4C_A, 2GHI_A, 5MKK_B, 4Q9H_A, 4AYW_A, 3VX4_A, 5U1D_B, 5U1D_A, 4PL0_A, 5L22_B, 4U00_A, 4K8O_A, 4MKI_B, 4HUQ_B, 3TUJ_C, 5NIK_J, and 5JSZ_A. Structural alignment and image rendering were carried out using ENDscript and PyMOL (See details in the Methods section)