Protein structure and function underpins all aspects of life from the structural framework of cells, enzymatic activity, cellular transport, to immune function. The production of the individual proteins is controlled through the expression of the corresponding genes. Riboswitches are non-coding RNAs mediating the regulation of genes by initiating or terminating transcription or translation. Here, gene expression is being triggered by environmental factors such as temperature or pH or through ligands like ions or small molecules. Ion or metabolite sensing riboswitches form highly selective binding pockets for their specific target. Fluoride sensing riboswitches are present in both, bacteria and archaea. Increasing F -concentrations can inhibit cell growth and become acutely toxic to cells. This antimicrobial effect has been exploited to eliminate harmful microorganisms in fermentation processes. Fluoride riboswitches are involved in cellular defence mechanisms, such as initiating the production of F -exporters. The first fluoride sensing riboswitch was found in the crcB motif of Pseudomonas syringae in 2012. In the same year, the crystal structure of the sensing domain of the fluoride sensing riboswitch from the bacterium Thermotoga petrophila in its F --bound form was reported by Ren et al. (Fig. ). The tertiary structure of the aptamer comprises two stems, a pseudoknot, and reversed Watson-Crick (A6•U38) and Hoogsteen (A40•U48) base pairs (Fig. ). The riboswitch coordinates a cluster of three Mg 2+ ions which in turn encapsulates the F -ion (Fig. ). In this way the polyanionic RNA avoids electrostatic repulsion with the bound F -. This motif composed of Mg 2+ , F -, phosphate, and water was computationally found to be stable largely based on the electrostatic interactions between F -and Mg 2+ . A recent computational study proposes a stepwise assembly of the cluster with two Mg 2+ bound in the apo form but the third Mg 2+ only being incorporated together with F -. The riboswitch is strongly selective for F -discriminating against other halides. Using solution nuclear magnetic resonance (NMR) spectroscopy, the fluoride binding riboswitch from Bacillus cereus was shown to adopt a highly similar fold. Here, stem 1 and stem 2 form already in absence of Mg 2+ and F -, whereas the formation of the pseudoknot requires presence of Mg 2+ . Further addition of F -does not result in a significant change of the tertiary structure but suppresses dynamics involving a lowly populated excited apo state responsible for transcription termination. Pulsed electron-electron double resonance (PELDOR, aka DEER for double electron-electron resonance ) spectroscopy permits measuring distances ranging from 20 Å to 100 Å and beyond between paramagnetic centres such as spin labels, paramagnetic metal ions, amino acid radicals and radical cofactors. This provides a unique opportunity for investigating conformational ensembles through their spin-spin distance distributions. Complementary to PELDOR, F electron-nuclear double resonance (ENDOR) is an emerging technique to detect distances in a shorter range of about 5 -20 Å between nitroxide spin labels and F nuclei. This has recently been expanded to distances between fluorine nuclei and other spin centres such as triarylmethyl (TAM) radicals, tyrosyl radicals, Cu II , or Gd III . In this work, we have investigated the T. petrophila riboswitch in solution by magnetic resonance methods. H NMR spectroscopy allowed monitoring the formation of base pairs through signals in the imino region of the free RNA, the Mg 2+ -bound apo riboswitch, and the F --bound holo riboswitch. We have further studied the solution-state preorganisation of the tertiary fold in the free and apo riboswitches by PELDOR and we have employed F ENDOR to investigate the structure of the binding pocket of the holo riboswitch. 4ENC) with the F -(green sphere) encapsulated by three Mg 2+ (blue spheres); (b) schematic representation of the secondary structure of the 50 nucleotide construct of the fluoride binding riboswitch used here, with the two stem structures (green and blue), the pseudoknot structure (magenta), and the reversed Watson-Crick (A6•U38) and reversed Hoogsteen (A40•U48) base pairs (salmon). Residue numbers for spin-labelling sites are indicated. The residues whose O -are involved in the coordination of the Mg 2+ are highlighted with red asterisks; (c) Graphical representation of the binding pocket of the fluoride binding riboswitch (PDB: 4ENC) with the F -(green sphere) encapsulated by three Mg 2+ (blue spheres). O -involved in the coordination are represented as red spheres. Additional ions not involved in the fluoride encapsulation and water molecules have been omitted for clarity throughout.