Pulsed ESR measurements were performed on a Bruker Elexsys E580 Q-Band spectrometer equipped with an arbitrary waveform generator (SpinJet-AWG), a continuous-flow helium cryostat, a temperature control system (Oxford Instruments), a 50 W solid state amplifier, and a Bruker EN5107D2 dielectric resonator. Field-swept (echo-detected) spectrum and phase memory time (𝑇 𝑀 ) and were acquired at 10 K for Gd 3+ or at 50 K for MTSL using the two pulse Hahn echo sequence 𝜋 2 -𝜏 -𝜋 ⁄ -𝜏 employing Gaussian pulses (32 ns or 48 ns, respectively) and an inter-pulse delay (𝜏) of 400 ns. The values for 𝑇 𝑀 and were estimated as the time corresponding to the decay of the signal by 1/e. PELDOR experiments were recorded using a dead-time free sequence using a Gaussian pump and observer pulses with a 16-step phase cycling to filter unwanted echoes (x[x][xp]x). For MTSL, data was acquired at 50 K using a 38 ns pump pulse and 48 ns observer pulses. The pump pulse was set to the maximum of the echo-detected field-swept spectrum, and the observer pulses were set at 80 MHz lower with a shot repetition time (SRT) of 2 ms. For nitroxide -Gd 3+ experiments, the temperature was set to 10 K to observe Gd 3+ while pumping nitroxide. A 24 ns pump pulse was used with 32 ns observer pulses, which were set at 280 MHz lower, and an SRT of 1 ms was used. For Gd 3+ -Gd 3+ experiments, the temperature was set to 10 K with an SRT of 1 ms. A 32 ns pump pulse was used at the spectral maximum with 32 ns observer pulses at 100 MHz lower. The deuterium modulations were averaged by increasing the first inter pulse delay by 16 ns for eight steps. Distance distributions were independently determined using the DeerLab program using a fractional dimensionality (d) for the spin distribution over the membrane surface (which gave d = ~2.2 for all the samples). The primary data were simultaneously fitted for the distances and the background function using a model-free approach. Simulations of the distance distributions were obtained on the structures using the rotamer libraries as implemented in the MATLAB-based software package MMM2022.2. Figure S2. (A) Structure of cyanocobalamin bound BtuF (PDB ID: 1N4A). Rotamers (in violet) for M-DOTA-Gd 3+ at position S138C and TEMPO labeled cyanocobalamin calculated using the MMM program are shown in the space-filling model. The size of the spheres corresponds to the relative probability for each rotamer. (B) PELDOR/DEER measurements between BtuFGd 3+ and TEMPO-cyanocobalamin. The primary data is shown on the left, with the determined distances on the right. The corresponding simulation is overlaid in red dotted lines. The TEMPOcyanocobalamin has a 70% labeling efficiency. The data yielded a 20% modulation depth. With a maximum modulation depth of ~30% under our experimental setup, it shows that BtuF is nearly 100% Gd 3+ labeled. The corresponding experimental data (please see Figure for more details) in the envelope and the simulations (in dotted black or orange lines) are shown in panel (C). An additional simulation (marked as 'Any rotamers?' as implemented in the MMM program) was performed (in solid black or orange lines) by allowing the rotamers to freely reorient to match the experimental distances to the best possible. These simulations show that the discrepancy could be accommodated through the rotamer redistribution to a large extent, though not completely. (B) BtuF, as it was observed in the above BtuCD-F apo structure, was placed above the BtuCD apo structure (PDB ID: 1L7V) through a global alignment using Pymol (no energy minimization was performed on this structure). BtuCD-F and BtuCD apo structures align with an overall RMSD of 1.25 Å, and the differences between TM5-TM5a helices (which are more open as highlighted in magenta) constitute one of the major differences. No steric clashes are apparent in this configuration. The interspin distance simulations and the corresponding experimental data for BtuCD-F + VitB12 (please see Figure for more details) in the envelope are shown in panel (D). The increase in experimental distances with VitB12 is in line with the simulations, suggesting that the VitB12 bound structure may have a similar conformation. Further supporting this notion, an additional simulation in which the rotamers are allowed to freely reorient shows a better match with the experimental distances.