The root primordial sequence was constructed using the marginal reconstruction algorithm. Superimpostion using Chimera We loaded chains F and G (MalF and MalG of the maltose transporter from E. coli K12) from PDB (# 2R6G) into UCSF Chimera 1.7 (http://www.cgl.ucsf.edu/chimera/). Initial TMS predictions
were taken from TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM/), and compared with the Protein Feature View at (http://www.rcsb.org/pdb/explore/explore.do?structureId=2R6G) for the F and G chains. The following approximate positions of the TMSs were used. MalF: 20–40; 40–60; 70–90; gap; 280–300; 320–340; 370–390; 430–450; 490–510. MalG: 20–40; 90–110; 120–140; middle; 155–175; 210–230; 260–280. The actual PDB file was downloaded and edited, so that it only
contained the lines starting with “ATOM”. We cut out the last 3 3 MA TMSs from each chain (MalF 360–504 and MalG 145–290) and transferred these to a new location. Motif Avapritinib chemical structure identification To search for matching segments between MalF and MalG, we blasted the sequence pair against each other and identified a motif, “EA + A + DGA”, located between TMS 1 and TMS 2 in the last 3 TMS segments of both MalF and MalG. We also identified other motifs, including “FPL+”, “+AI”, “SW”, and “DxW+LAL”. To confirm the hypothesis that it is TMSs 3, 4 and 5 in MalF that correspond to TMSs 1, 2 and 3 in MalG, we extracted the following atom coordinate sets from the “”2R6G”" model: 65 – 350 in MalF and 10 – 150 in MalG. These alpha carbon traces were Ketotifen superposed in Chimera in the same way as previously described. Ancient Rep To compare our results using Protocol 1 and Protocol 2, we focused on the last 3 TMSs in MalF and MalG. These sequences have a common fold, but the sequence similarity is not PI3K Inhibitor Library concentration apparent. We took sequences from LFG … KFD in MalF, and sequence from IPF … to VKG in
MalG. These were entered into Protocol 1 , setting CD-HIT to 0.8. In Protocol 2, the best scoring pair for the comparison of two lists of hits from an iterative search based on the last 3 TMSs in MalF and MalG, had a GSAT Z-score of 21 S.D., far in excess of what is required to establish homology. Protocols 1 and 2 are standard tools, part of the BioV Suite, reported by Reddy and Saier (2012). Protocol 1 runs a PSI-BLAST search with iterations, collects results, removes redundant/similar sequences, annotates, tabulates, and counts TMSs. Protocol 2 allows the rapid identification of homologs between any two FASTA files using the G-SAT program also described by Reddy and Saier . To elucidate the domain duplication history of MalG, we ran Protocol 1 on MalG in preparation for running ANCIENT REP . We took P68183 from http://www.tcdb.org/search/result.php?tc=3.A.1.1.1, not counting TMSs, using “test” as the output path, and 0.8 as the CD-HIT threshold. We then used “ancient -i results.faa -r 3 -o test2 –method = 3 –threads = 4”. We repeated for MalF.