Fig. 6: Cryo-EM structure of BtuB3G3-CNcbl and functional analysis.

a Cryo-EM map for BtuB3G3-CNCbl state 1. BtuB3 is yellow, BtuG3 blue and CNCbl pink. b Cartoon superposition showing BtuB3G3-CNCbl state one (blue and yellow, vitamin in magenta) and BtuG3-CNCbl (wheat, vitamin in grey). c Close-up of the residues involved in vitamin binding by BtuG3 and BtuB3G3. Note that Y538 in EL8 (yellow) is the only BtuB3 residue that contacts the CNCbl molecule. d Top panel, superposition of BtuB3G3-CNCbl states 1 (wheat) and 2 (pale green). Bottom panel, close up view highlighting the differences in EL8. For clarity, loops are smoothened. The red asterisks denote the missing part of EL8 in state 2. e Same as d, but including the crystal structure of BtuB2G2 (in blue and yellow). Middle and right panel show close up views of EL8 and CNCbl. Note how CNCbl for BtuB3G3 state 2 (green) is slightly displaced towards BtuB3 relative to state 1. f Growth curves for WT (loci 1 and 2 deleted and a hexa-histidine tag in btub3) and EL8 mutant (WT plus the EL8 deletion; ΔEL8) strains in methionine, B₁₂-replete (40 nM CNCbl) and B₁₂-limiting conditions (0.4 nM CNCbl). Data are representative of three independent trials; error bars indicate ±SD from three technical replicates. Source data are provided. g Representative SDS-PAGE gel (n = 2 independent experiments) showing purified BtuB3G3 and IMAC elutions from WT and ΔEL8 strains. Note that the non-boiled samples show a stable complex, which after boiling dissociates in BtuB3 and BtuG3 (uncropped gels in Source Data).