Fig. 1.

Function and structure of BtuM_Td. a Schematic representation of cobalamin (Cbl) showing the corrinoid ring with the central cobalt ion (red). The ligand at the β-axial position is in this case a cyano-group, but differs in various Cbl variants (Supplementary Figure 1a, b). The ligand at the α-axial position (base-on conformation) is the 5,6-dimethylbenzimidazole base, which is covalently linked to the corrinoid ring. When this coordination is lost, Cbl is termed base-off. Cbi lacks the 5,6-dimethylbenzimidazole base (indicated by the zigzagged red line). b Growth assays with E. coli ΔFEC was conducted in the presence of 50 μg ml⁻¹ l-methionine or 1 nM Cbl. Additional experiments in the presence of different Cbl concentration are shown in Supplementary Figure 2f and g. All growth curves are averages of nine experiments (three biological triplicates, each with three technical replicates). Top panel: cells containing the empty expression vector (pBAD24) in the presence of methionine (blue line) or Cbl (grey line) and cells expressing the BtuCDF system (black and red lines, respectively). Bottom panel: cells expressing BtuM_Td (black and red lines) or mutant BtuM_Td_C80S (blue and grey) in the presence of methionine and Cbl, respectively. The inset displays a western blot showing that the mutant is expressed to wild-type levels (the full-length western blot can be found in Supplementary Figure 2h). c The structure of BtuM_Td in cartoon representation, coloured from blue (N terminus) to red (C terminus) and viewed from the membrane plane. α-helices (H1-6) and connecting loops (L1-5) are indicated. Cbl is shown in stick representation with carbon atoms coloured wheat, the oxygen and nitrogen atoms in red and blue, respectively, the cobalt ion in pink. Four n-nonyl-β-d-glucopyranoside detergent molecules are also shown in stick representation (carbons in light grey)