Structural conservation was calculated and visualized using ENDscript/ESPript v. 3.0  with the PDBAA95 database, E-value of 1e-12, and contact range of 2.7 Å. COACH  was used to predict ligand-binding sites in CsMRP4. SDF files for bile acids were retrieved from the PubChem database  as of August 2017 and transformed into the MOL2 format using OpenBabel . Bile acids were docked into CsMRP4 using PyRx v. 0.8 , which includes AutoGrid  and AutoDock Vina . A grid box extended to all membrane-spanning domains (MSDs) of CsMRP4; no information regarding the exact location of the binding sites of the various bile acids was available. Active site dimensions were set as the grid size of center X: 25.6 Å, center Y: -13.5 Å, and center Z: -6.5 Å, and 8 maximum exhaustiveness was calculated for each bile acid. All structure visualizations were carried out using UCSF CHIMERA v. 1.10.2  and PyMOL Molecular Graphics System v. 1.7.4.5 (Schrödinger, LLC., New York, NY, USA).