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Distance measurements by pulse electron paramagnetic resonance (EPR) techniques, especially the double electron-electron resonance (DEER) experiment, are an established procedure that is extensively used with nitroxide spin pairs for structure determination studies and the observation of structural changes of bio-macromolecules. Recently, it was increasingly applied to multiple-spin systems. In the DEER experiment, more than two dipolar coupled spins manifest in an increased total modulation depth and in sum and di erence dipolar frequency contributions that give rise to additional peaks appearing in the distance distribution, which do not correspond to the real interspin distances of the system and are hence referred to as ghost contributions. These ghost contributions may be so prominent that they might be mistaken for real distance peaks or that real distance peaks shift their position or disappear. While the position of ghost distances is determined by the interspin distance geometry, their prominence and extent in the DEER form factor is determined by the inversion e ciency. A precise evaluation of the inversion e ciency in order to estimate the magnitude of ghost contributions is therefore a prerequisite for a precise and accurate analysis of multi-spin distance distributions. Closely connected to the precise determination of inversion e ciency is the precise determination of total modulation depth. The total modulation depth increases with increasing spin multiplicity N . The form and dimension of the background decay crucially determines the numerical value of total modulation depth. Exact determination of the background decay is furthermore needed for the separation of interand intramolecular contributions. The exact form of the background decay depends on interpulse delay time and concentration and size of paramagnetic centers. For a given sample, spectrometer and probehead, the background decay is very well reproducible. Fitting the background dimensionality during data analysis is a simple way of tting most e ects that in uence the background decay and gives reasonable and reproducible values of modulation depth. The inversion e ciency can reliably be back-calculated from the total modulation depth ∆ when the spin multiplicity N is known. The linear dependency between the experimental inversion e ciency λexper of the echo-detected nutation experiment and the inversion

Quantifcation of Modulation Depth and Ghost Distance Removal in Dipolar EPR on Multiple-Spin Systems