Caused by a variety of over 100 different point mutations in mitochondrial tRNA genes, these diseases share a respiratory deficiency, caused by insufficient levels of functional tRNAs in mitochondrial protein synthesis. We strive to show that most of the tRNA deficiencies result from incomplete tRNA modification. Our greatest achievement up to date is the demonstration of the structural influence of a mitochondrial tRNA modification by single-molecule FRET. We could demonstrate that a single methylation shifts a dynamic equilibrium towards the functional cloverleaf tRNA structure. We are now in a position to assess the effect of pathogenic mutations, as well as that of protein binding to tRNA by smFRET. These experiments are paralleled by the related biochemistry and cell biology investigations in cybrid cell lines, which contain homoplasmic mutant mitochondrial DNA. The smFRET work in particular produced a strong resonance in the community, resulting in numerous invitations to conferences and secondary literature contributions.