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In this work, the experimental conditions and parameters necessary to optimize the long-distance (≥ 60 Å) Double Electron-Electron Resonance (DEER) measurements of biomacromolecules labeled with Gd(III) tags are analyzed. The specific parameters discussed are the temperature, microwave band, the separation between the pumping and observation frequencies, pulse train repetition rate, pulse durations and pulse positioning in the electron paramagnetic resonance spectrum. It was found that: (i) in optimized DEER measurements, the observation pulses have to be applied at the maximum of the EPR spectrum; (ii) the optimal temperature range for K a -band measurements is 14-17 K, while in W-band the optimal temperatures are between 6-9 K; (iii) W-band is preferable to K a -band for DEER measurements. Recent achievements and the conditions necessary for short-distance measurements (&lt;15 Å) are also briefly discussed.

Optimization of Pulsed-DEER Measurements for Gd-Based Labels: Choice of Operational Frequencies, Pulse Durations and Positions, and Temperature