Distance distribution information obtained by pulsed dipolar EPR spectroscopy provides an important contribution to many studies in structural biology. Increasingly, such information is used in integrative structural modeling, where it delivers unique restraints on the width of conformational ensembles. In order to ensure reliability of the structural models and of biological conclusions, we herein define quality standards for sample preparation and characterization, for measurements of distributed dipole–dipole couplings between paramagnetic labels, for conversion of the primary time-domain data into distance distributions, for interpreting these distributions, and for reporting results. These guidelines are substantiated by a multi-laboratory benchmark study and by analysis of data sets with known distance distribution ground truth. The study and the guidelines focus on proteins labeled with nitroxides and on double electron–electron resonance (DEER aka PELDOR) measurements and provide suggestions on how to proceed analogously in other cases.

Methods for measuring nanometer scale distances between specific sites in biomolecules (proteins and nucleic acids) and their complexes are essential for describing and analyzing their structure and function. In the last decade pulse EPR techniques were proven very effective for measuring distances between two spin labels attached to a biomolecule. The most commonly used spin labels for such measurements are nitroxide stable radicals. Recently, a new family of spin labels, based on Gd³⁺ chelates, has been introduced to overcome some of the limitations of using nitroxides, particularly at high magnetic fields, which are attractive due to the increased sensitivity they offer. The benefits that such S = 7/2 spin labels offer for frequencies of 30 GHz and higher, particularly at 95 GHz, include (1) high sensitivity, only ∼0.15 nmol of doubly labeled biomolecule is needed, (2) the lack of orientation selection, which allows straightforward data analysis. Gd³⁺–Gd³⁺ DEER (double electron–electron resonance) distance measurements on labeled peptides, proteins and DNA have already been demonstrated and the results show that they are very promising in terms of sensitivity. In this Perspective we review these new developments. We briefly introduce the characteristics of the DEER experiment on a pair of S = 1/2 spins and characterize the EPR spectroscopic properties of Gd³⁺ ions. We then introduce some of the tags employed to attach Gd³⁺ to biomolecules and provide a few experimental examples of Gd³⁺–Gd³⁺ DEER measurements. This is followed by a discussion of the parameters that affect the sensitivity of such DEER measurements. Since an important term in the spin Hamiltonian of Gd³⁺ is the zero-field splitting (ZFS), its effect on the DEER modulation frequencies must be considered and this is discussed next. Finally, another recently reported approach for using Gd³⁺ in distance measurements will be presented: the use of Gd³⁺–nitroxide pairs.

Gd(III) complexes have emerged as spin labels for distance determination in biomolecules through double-electron–electron resonance (DEER) measurements at high fields. For data analysis, the standard approach developed for a pair of weakly coupled spins with S = 1/2 was applied, ignoring the actual properties of Gd(III) ions, i.e. S = 7/2 and ZFS (zero field splitting) ≠ 0. The present study reports on a careful investigation on the consequences of this approach, together with the range of distances accessible by DEER with Gd(III) complexes as spin labels. The experiments were performed on a series of specifically designed and synthesized Gd-rulers (Gd-PyMTA–spacer–Gd-PyMTA) covering Gd–Gd distances of 2–8 nm. These were dissolved in D₂O–glycerol-d₈ (0.03–0.10 mM solutions) which is the solvent used for the corresponding experiments on biomolecules. Q- and W-band DEER measurements, followed by data analysis using the standard data analysis approach, used for S = 1/2 pairs gave the distance–distribution curves, of which the absolute maxima agreed very well with the expected distances. However, in the case of the short distances of 2.1 and 2.9 nm, the distance distributions revealed additional peaks. These are a consequence of neglecting the pseudo-secular term in the dipolar Hamiltonian during the data analysis, as is outlined in a theoretical treatment. At distances of 3.4 nm and above, disregarding the pseudo-secular term leads to a broadening of a maximum of 0.4 nm of the distance–distribution curves at half height. Overall, the distances of up to 8.3 nm were determined, and the long evolution time of 16 μs at 10 K indicates that a distance of up to 9.4 nm can be accessed. A large distribution of the ZFS parameter, D, as is found for most Gd(III) complexes in a frozen solution, is crucial for the application of Gd(III) complexes as spin labels for distance determination via Gd(III)–Gd(III) DEER, especially for short distances. The larger ZFS of Gd-PyMTA, in comparison to that of Gd-DOTA, makes Gd-PyMTA a better label for short distances.