The left panels illustrate the characteristic properties probed by the experiments: (A) small angle X-ray scattering (SAXS), (B) double electron-electron resonance spectroscopy (DEER), and (C) Förster resonance energy transfer spectroscopy (FRET). In general, all middle panels display representations of the experimental data (dark yellow curves). The right panels show model-free analysis (red). Predicted experimental data based on a full-length X-ray crystal structure (PDB-ID: 1DG3) are shown in blue. To the top of the experimental curves, either data noise weighted, w.res., or unweighted residuals, res., are shown (middle panels). DEER and FRET experiments sense distances between labels that are flexibly coupled to specific labeling sites (exemplified for the double cysteine variant Q344C/A496C). The time-dependent responses of the sample (middle) inform on the inter-label distance distributions (right panels). The recovered distance distributions are compared to structural models by simulating the spatial distribution of the labels around their attachment point (left panels). The spatial distributions of the MTSSL-labels (B, left), as well as the donor and acceptor dye (C, left), are shown in magenta, green, and red, respectively. All distances resolved by EPR and FRET are compiled in Appendix 1—table 1 (A) Left: In SAXS the scattered intensity I(q) is measured as a function of the scattering vector q. Middle: For better illustration, I(q) is presented in a Kratky-plot. The data are deposited in SASBDB (ID: SASDDD6). Right: SAXS ab initio bead modeling determines an average shape of hGBP1 in solution. (B) Left: The DEER experiments measured the dipolar coupling between two MTSSL spin-labels (magenta). Middle: DEER-traces, F(t), analyzed by Tikhonov regularization (red curve). Right: Recovered inter-spin distance distributions, p(RSS). (C) Left: FRET experiments measure the energy transfer from a donor fluorophore (Alexa488, green) to an acceptor fluorophore (Alexa647, red). Middle: Fluorescence intensity decays of the donor analyzed by the maximum entropy method (MEM) recover donor-acceptor distance distributions, p(RDA). The inset displays the L-curve criterion of the MEM reconstruction for the presented data set. The FRET-induced donor decay, εD(t), represents the fluorescence decays (Peulen et al., 2017). εD(t) is corrected for the fraction of FRET-inactive molecules, xDOnly. The shape of εD(t) reveals characteristic times (labeled M1 and M2) that correspond to peaks in p(RDA). Right: Recovered inter-label distance distributions for FRET.