A segment of mouse NBCe1-B, consisting of 1000 amino acids (NP_061230.2), was submitted to both ROBETTA and hhPRED. The ROBETTA software generated different models for three segments of the submitted sequence. No homology was identified for segment 1-106. Segment 107-413 was homologous to Putative Substrate Access Tunnel in the Cytosolic Domain of Human Anion Exchanger 1 (Protein Data Bank ID: 4KY9), which is similar to human erythrocyte band 3 cytoplasmic domain (Protein Data Bank ID: 1HYN) that we previously used to predict NBCe1-B N terminus (16), and the same structure is used here. The transmembrane segment spanning residues 414 to 1000 was found to be homologous to the structure of the anion exchanger domain of human erythrocyte band 3 (Protein Data Bank ID: 4YZF). The highest transmembrane domain (TMD) homology score provided by the hhPRED software was for the Electrogenic sodium bicarbonate cotransporter 1, NBCe1 (PDB_ID 6CAA). (Probability, 100; E value, 2.5 × 10−164; SS,108.9; Cols,1003). The NBCe1 TMD structure has been recently solved by cryo–electron microscopy (CryoEM) (41). The final model was generated with PyMOL using the TMD obtained of the CryoEM structure, and the intracellular domains were predicted by hhPRED and ROBETTA based on the N-terminal domain of AE1. The orientation of the N terminus with respect to the TMD is not known with certainty and may not be accurate. However, the overall structure is reasonable because the software identified the N terminus of AE1 as the best template, which, like NBCe1-B, is a member of the SLC4 transporters superfamily. In addition, we have previously predicted the structure of NBCe1-B to be similar to the crystal structure of UraA (16), which is similar to the crystal structure of AE1. This indicates that the software predicted the same structure twice and independently on the basis of several similar, yet different structures.

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