All constructs were designed based on the crystal structure of the T. petrophila riboswitch. The labelling sites were screened using in silico labelling with MtsslSuite and MMM to predict distances between pairs of spin labels or spin labels and F -. Furthermore, ensembles of spin label rotamers can also be extracted allowing analysis of orientation selection in the anisotropic EPR spectrum. In absence of an experimental highresolution structure a similar modelling can be applied using computational structure predictions that have become available also for nucleic acids with the introduction of AlphaFold3. To select the labelling sites, following general criteria were applied: (i) nucleotides involved in the formation of the cluster, i.e. residues coordinating Mg 2+ were excluded (red asterisks in Fig. ); (ii) distances in the range between 18 -45 Å were considered for PELDOR, and distances below 20 Å for F ENDOR.