Notably, the BtuF-Gd 3+ -BtuC-NO PELDOR revealed distinct modulation and interspin distances (Figure ; please see methods for details of sample preparation). As expected, a similar experiment without BtuCD overexpression did not reveal any distances (Figure ), thereby confirming that the observed distances arise exclusively from BtuC-BtuF interaction. Another Gd 3+ -Gd 3+ experiment on this sample gave only an exponential decay, showing that the BtuF molecules are far separated and do not interact within the envelope. The experimental distances are longer than the corresponding simulations (Figure ), which might be explained either with a different rotamer distribution and or a somewhat increased flexibility at the BtuF-BtuC interface (Figure ). The distance distribution determined using the DeerLab program for the 15 The modulation depth () of the PELDOR data provides quantitative information on the extent of BtuF-BtuC interaction. The  is the highest at the point of maximal BtuF-BtuC interaction, and as the fraction of unbound BtuF increases, they  will decrease. We observed the maximum  (~13%) with 10 M BtuF, and it further decreased at higher concentrations, thereby confirming the specific interaction between BtuF and BtuC (Figure ). Considering the sub-nanomolar affinity between BtuF and BtuCD, this interaction must already be saturated at 10 M. With max = ~30% under our experimental setup for a twospin system, it would further increase for a three-spin system (up to ~50%) if both BtuF and BtuC are 100% labeled (BtuF has a ~100% L.E., Figure ). The reduced modulation depth we observed could arise if the amount of BtuC is lower than the added BtuF (10 M) and or due to a lower labeling efficiency for BtuC in the envelope environment (the reality might be a combination of both). Provided that sufficient S/N is achieved, a lower  for a multi-spin system helps to eliminate the undesired artifacts. In summary, the tested conditions allow for reliable PELDOR experiments with BtuCD-F in the native envelope. as well are overlaid.