The cysteine variants of BtuF and BtuCD were created using the QuickChange site-directed mutagenesis kit (Agilent) according to the protocol of the manufacturer. For expression of BtuF S138C, a construct with a 6xHis tag fused to the C-terminal region was transformed into E. coli BL21-CodonPlus (DE3)-RIPL competent cells (Agilent, 230280). A 100 mL of TB medium supplemented with 100 µg/mL ampicillin was inoculated with a single colony, and cells were grown overnight at 33°C. A 10 mL of the overnight culture was used to inoculate 1 L of TB medium containing 100 µg/mL ampicillin. The cells were cultured at 37 °C, induced with 0.4 mM IPTG at an OD600 of 0.7, and further grown at 25 °C for 4 h. For BtuCD, the construct contained an N-terminal 10x His-tag fused to BtuC. To prepare pre-culture, TB medium supplemented with 100 µg/mL ampicillin was inoculated with a single transformed colony of BL21 Star (DE3)pLysS, and cells were grown overnight at 30 °C. A 5-10 mL of the pre-culture was used to inoculate 1.3 L of TB medium containing 100 µg/mL ampicillin. The cells were cultured at 37 °C, induced with 0.4 mM IPTG at an OD600 of 0.7, and further grown at 25 °C for 4 h. In both cases, cells were collected by centrifugation at 12,000xg and stored at -80°C for further use.