Fig. 5. MenA1 and MenT1 form an asymmetric heterotrimeric complex.

A 1 µg total RNA of M. smegmatis was incubated with MenT1 (5 µM), along with [α-³²P]-labelled CTP, at 37 °C for 2 h, in the presence of increasing molar ratios of MenA1 antitoxin. Representative results of triplicate experiments are shown. B [α-³²P]-CTP labelled M. tuberculosis tRNA Gly-3 was incubated with 5 µM of MenT1 WT or MenT1 D41A at 37 °C for 4 h in the presence of unlabelled CTP and increasing molar ratios of MenA1 antitoxin. Representative results of triplicate experiments are shown. Source data are provided as a Source data file. C Purified MenT1 and MenA1 samples were combined and incubated overnight, then separated by Size Exclusion Chromatography (SEC) using a HiPrep 16/60 Sephacryl S-200 SEC column (Cytiva). MenAT1 eluted at an elution volume of 52.7 ml, corresponding to a mass matching the predicted Mr of a MenT1:MenA1:MenT1 heterotrimeric complex. MenT1 eluted at 63.4 ml, corresponding to a mass matching monomeric MenT1. D, E Structure of the MenAT1 complex, with views from each side, above and below, shown as cartoons coloured orange (MenT1α), blue (MenA1), and light orange (MenT1β). N and C termini are indicated. To help compare their relative positions versus MenA1 the locations of active site residues identified in Fig. 2F (T39, D41, K137 and D152) are indicated, and the residues are shown as sticks, coloured red for oxygen, blue for nitrogen. Black boxes drawn in (E) correspond to regions shown in detail as panels Fig. 6A–D.