Nucleic Acids Research, 2012, Vol. 40, Database issue D365 Figure 2. (A) Superposition of the modeled MIP structure with 1J4N, the structure of bovine aquporin shown in two different orientations, namely, parallel (left) and perpendicular (right) to the channel axis. (B) The four residues of the ar/R selectivity ﬁlter superposed on that of 1J4N structure. (C) Comparison of HOLE radius proﬁles plotted for the water channel (green), glycerol facilitator (blue) and the modeled MIP structure (red). The ar/R selectivity ﬁlter is approximately located at 10 A˚ . (D) Phylogenetic tree calculated for all MIPs of a representative organism Phytophthora infestans using parsimony method. segments and loop E. This aromatic/arginine (ar/R) select- ivity ﬁlter has been implicated in obstructing the proton conduction (62) and efﬁcient solute transport (37,63,64). The four ar/R selectivity ﬁlter residues, represented by their one letter codes, are given for each MIP. For example, ‘FIIR’ indicates that the ﬁrst and second residues Phe and Ile are from TM2 and TM5, respectively, and the last two are the loop E residues. Analysis of selectivity ﬁlter residues indicates that there are 349 MIPs, which have se- lectivity ﬁlter (FHTR, FHAR, FHCR or FHSR) similar to that found in water-selective aquaporin channels (FHTR or FHCR). The number of MIPs having selectiv- ity ﬁlter (WGYR, WGFR or WGWR) similar to the glycerol-speciﬁc aquaglyceroporin (WGFR) is 170. Thus, about 50% of the total MIPs in the database have select- ivity ﬁlter typical of aquaporin or aquaglyceroporin. The remaining half has substitutions that can alter the size and chemical nature of the outer constriction. This will have major inﬂuence in the nature of solute that is being trans- ported by the channel. The channel diameters of water channel and the aquaglyceroporin at the ar/R constriction are 2.0 and 3.5 A˚ , respectively. The selectivity ﬁlter residues of the predicted MIP model superposed on that of the pure water channel from bovine aquaporin and that of aquaglyceroporin from E. coli are available (Figure 2B). The HOLE radius proﬁles of all three MIPs, water channel, glycerol channel and the modeled MIP channel, can be compared (Figure 2C). This will give an idea about the size of the region around the ar/R se- lectivity ﬁlter region with respect to both water channel and aquaglyceroporin and it will help the user to predict the possible size of a solute that can pass through this constriction. Structure-based sequence alignment As mentioned earlier, MIP sequences are diverse. For example, the sequence identities between some of the plant MIP subfamilies are as low as 20% (13,15). In such cases, programs such as ClustalW (65) that are used to generate multiple sequence alignment of a given set of sequences are unlikely to produce meaningful align- ment for a diverse set of MIP sequences. Instead, if the structurally equivalent positions belonging to the TM helical segments are aligned, they are likely to result in high conservation and indicate the importance of residues in certain positions. Structure-based sequence alignment