## 2 Results
### Validation of PTS-Pre Prediction Using Experimentally Determined trans-PTs Sequences
There was only one sequence each in Chlamydia and Chlorobi, but neither of them was predicted correctly. More incorrect cases were found in Deinococcus-Thermus, Actinobacteria, Firmicutes, and Proteobacteria with prediction accuracies of 33% (1/3), 71% (10/14), 80% (12/15), and 88% (35/40), respectively (Figure S4B). Taken together, 85% (17/20) of total incorrectly predicted cases come from these bacterial phyla, which suggests that the prediction accuracy may be closely related to the species. Figure 3: The prediction and verification results of PTS-Pre. (**A**) Comparison of prediction accuracies between MD-Pre and PTS-Pre over 74 testing sequences. (**B**) Comparison of prediction accuracies across three life kingdoms. Panels with comparatively higher or lower accuracy are marked with asterisks. (**C**) Comparison of prediction accuracies using different searching templates. Panels with comparatively higher or lower accuracy are marked with asterisks. In (**A–C**), the quantities of correctly predicted and total cases are labeled on the top of each panel. (**D**) In vitro activity of predicted _trans_-PTs from _Streptomyces azureus, Topvothrix camylomenoides_, and _Gossypium arboreum_. All predicted _trans_-PTs were characterized using DMAPP and [1–14C]-IPP as co-substrates; the products were analyzed by TLC. AtGFPPS4 was used as a positive control. (**E**) The distribution of predicted products in the _trans_-PTs family. Consequently, we assigned all the testing sequences into the SSN map and compared the distribution of incorrect and correct predictions. As shown in Figure 2A, the testing sequences were distributed in most of the predominant clusters and dispersed randomly in the map, which was compatible as a testing dataset. Consistent with our hypothesis, the incorrect prediction cases were enriched in tiny clusters near the searching templates 3Q2Q (_Corynebacterium glutamicum_, Bacteria), 3 PKO (_Levilactobacillus brevis_, Bacteria), and 1 WMW (_Thermus thermophilus_, Bacteria), while the correct prediction cases were enriched in six main clusters with the searching templates 2 FOR (_Shigella flexneri_, Bacteria), 5E8H (_Arabidopsis thaliana_, Eukarya), 1 WYO (_Pyrococcus horikoshi_, Archaea), 2E8W (_Saccharomyces cerevisiae_, Eukarya), 3 OYR (_Caulobacter vibrioides_, Bacteria), 3 AQQ (_A. thaliana_, Eukarya), 3 QQV (_Corynebacterium glutamicum_, Bacteria), and 1 FPS (_Gallus gallus_, Eukarya). We further calculated the prediction accuracy for each of the searching templates, and the results show that the searching templates 1 FPS (95% accuracy, 19/20), 2 FOR (93% accuracy, 41/44), and 3 OYR (88% accuracy, 15/17) had extremely high accuracies, while 3Q2 QQ (60% accuracy, 3/5), 3 PKO (0% accuracy, 0/3), and 1 WMW (66% accuracy, 2/3) had lower accuracies (Figure 3C; the complete data are shown in Table S5). The accuracy for 3 PKO is unusual as none of the three sequences (GI 28377915, 116334218, and 29376566) which selected 3 PKO as the best template were correctly predicted. All three testing sequences belong to Firmicutes and synthesize products with chain lengths longer than C30 [14]. However, PTS-Pre predicted their activities as GGPPSs (C20) because the \"second floor\" of these proteins is blocked by two residues with large side-chains (these three proteins have the same \"second floor\" residues: site_1 'Leu', site_2 'Phe', site_3 'Met') although they can pass through the \"first floor\" (\"first floor\" residues: GI 28377915, site_1 'Ala', site_2 'Thr', site_3 'Ile'; GI 28377915, site_1 'Gly, site_2 'Thr', site_3 'Ile'; GI 28377915, site_1 'Ala', site_2 'Thr', site_3 'Leu') (Figure S5). The conflict between the experimental data and the \"three floors\" model may be explained by the side-chain orientation rearrangement of blocking residues, thus creating a new product chain elongation tunnel along the interface of the _trans_-PT homodimer.