DEER experiments were conducted to determine the distance between the spin labels in the doubly-labelled CaM/M13 complex. The presented data was acquired at 120 K, with distance distributions obtained through analysis with DEERNet (Figure ). Additional DEER experiments performed at 50 K and even 180 K, and distances obtained with DeerAnalysis, are available in the Supplementary Information. The bimodal distribution in Figure has maxima at 36 Å and 42 Å. The bimodality of the peak can be explained as a consequence of a complex landscape for the spin label positions with respect to the protein backbone. The more intense 36 Å peak coincides well with measurements of this protein using MTSL, a commonly used cysteine-selective, five-membered, tetramethyl-substituted spin label. The 42 Å distance coincides well with a simulated distance (chiLife) of this protein complex with the two cysteine mutations and labelled with MTSL. The overlay of the distance distribution from Figure with the simulated and experimental MTSL distances is shown in the Supplementary Information (Figure ).