  Relaxation-induced dipolar modulation enhancement (RIDME) is a pulse EPR
technique that is particularly suitable to determine distances between
paramagnetic centers with a broad EPR spectrum, e.g. metal-ion-based ones. As
far as high-spin systems (S > 1/2) are concerned, the RIDME experiment provides
not only the basic dipolar frequency but also its overtones, which complicates
the determination of interspin distances. An r.m.s.d.-based approach for the
calibration of the overtone coefficients is proposed and illustrated for a
series of molecular rulers doubly labelled with Gd(III)-PyMTA tags. The
constructed 2D total-penalty diagrams clearly show that there is no unique set
but rather a certain pool of overtone coefficients, which can be used to
extract distance distributions between high-spin paramagnetic centers as
determined from the RIDME experiment.
