Q-band DEER data and fit for T4L spin-labeled with MTSSL at residues 65 and 80. (A) The crystal structure of T4L (PDB accession no. 2LZM; Matsumura et al., 1989) showing the location of the two spin labels. pdb, Protein Data Bank. (B) The scaled DEER data are shown as filled black circles together with the best model-based fit (red line) and background factor (green line). (C) The best-fit P(R) modeled as the sum of two Gaussians is shown as a red line with the associated 2σ (95%) confidence band as a shaded region. The model-free P(R) obtained using Tikhonov regularization in DeerAnalysis 2019 is also shown (dotted blue line). The optimal regularization factor was determined by generalized cross-validation and the starting time point for background fitting using the validation tool. (D) The solid red line and shaded region duplicate those shown in C. The best-fit P(R) modeled as the sum of 2 Gaussians obtained from 15 replicate datasets are shown as thin black lines.