For DEER/PELDOR experiments, MTSL-labeled envelope samples containing overexpressed BtuCD were diluted to 100 -200 M spin. BtuF, VitB12, and or nucleotides were introduced into the envelope samples by three to four cycles of freeze and thaw in liquid nitrogen. F or samples containing the nucleotide, initially, the nucleotide alone was introduced into the envelope, followed by incubation at 37 °C for 5 min. Subsequently, BtuF and or VitB12 was introduced by additional freeze-thaw cycles. All samples containing 20% d8-glyerol were transferred into 1.6 mm outer diameter quartz ESR tubes (Suprasil, Wilmad LabGlass) and snap-frozen in liquid nitrogen.