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.

EPR-based nanometre distance measurements are becoming ever more important in structural biology. Usually the distance constraints are measured between two nitroxide spin labels. Yet, distance measurements between a metal center and spin labels enable, e.g., the localization of metal ions within the tertiary fold of biomolecules. Therefore, it is important to find methods that provide such distance information quickly, with high precision and reliability. In the present study, two methods, pulsed electron-electron double resonance (PELDOR) and relaxation-induced dipolar modulation enhancement (RIDME), are compared on the heme-containing and spin-labeled cytochrome P450cam. Special emphasis is put on the optimization of the dead-time free RIDME experiment and several ways of data analysis. It turned out that RIDME appears to be better suited for distance measurements involving metal ions like low-spin Fe(3+) than PELDOR.

Nanometer distance measurements based on electron paramagnetic resonance methods in combination with site-directed spin labelling are powerful tools for the structural analysis of macromolecules. The software package mtsslSuite provides scientists with a set of tools for the translation of experimental distance distributions into structural information. The package is based on the previously published mtsslWizard software for in silico spin labelling. The mtsslSuite includes a new version of MtsslWizard that has improved performance and now includes additional types of spin labels. Moreover, it contains applications for the trilateration of paramagnetic centres in biomolecules and for rigid-body docking of subdomains of macromolecular complexes. The mtsslSuite is tested on a number of challenging test cases and its strengths and weaknesses are evaluated.

Metal ions play an important role in the catalysis and folding of proteins and oligonucleotides. Their localization within the three-dimensional fold of such biomolecules is therefore an important goal in understanding structure-function relationships. A trilateration approach for the localization of metal ions by means of long-range distance measurements based on electron paramagnetic resonance (EPR) is introduced. The approach is tested on the Cu(2+) center of azurin, and factors affecting the precision of the method are discussed.

A rigid, nitroxide substituted terpyridine ligand has been used to synthesize hetero- and homoleptic bis-terpyridine complexes of copper(II). The homoleptic complex represents a three-spin system, while the metal ion in the heteroleptic complex is in average bound to one nitroxide bearing ligand. Both complexes are used as model systems for EPR distance measurements using pulsed electron-electron double resonance (PELDOR or DEER) and relaxation induced dipolar modulation enhancement (RIDME) sequences. The results of both methods are analyzed using detailed geometric data obtained from the crystal structure of the homoleptic complex as well as information concerning ligand scrambling and the electronic structure of the copper center. In addition, both methods are compared with respect to their sensitivity, the extent of orientation selectivity and the influence of multispin effects.

Pulsed electron paramagnetic resonance (EPR) dipolar spectroscopy (PDS) offers several methods for measuring dipolar coupling constantsa nd thus the distance between electron spin centers. Up to now,P DS measurements have been mostly applied to spin centers whose g-anisotropies are moderatea nd therefore have an egligible effect on the dipolar coupling constants. In contrast, spin centers with large g-anisotropy yield dipolarc oupling constants that depend on the g-values.I nt his case, the usual methods of extracting distances from the raw PDSd ata cannot be applied. Here, the effect of the g-anisotropyo nP DS data is studied in detail on the example of the low-spin Fe 3 + ion. First, this effect is described theoretically,u sing the work of Bedilo andM aryasov (Appl. Magn. Reson. 2006,3 0, 683-702) as ab asis. Then, two knownF e 3 + /nitroxide compounds and one new Fe 3 + /trityl compound were synthesized and PDS measurements were carried out on them using am ethod called relaxation inducedd ipolarm odulation enhancement (RIDME). Based on the theoretical results, aR IDME data analysis procedure was developed, which facilitated the extraction of the inter-spin distance and the orientation of the inter-spin vector relative to the Fe 3 + g-tensor frame from the RIDME data. The accuracyo ft he determined distances and orientationsw as confirmed by comparison with MD simulations. This methodc an thus be appliedt ot he highly relevant class of metalloproteins with, for example, low-spin Fe 3 + ions.[a] Dr.Supporting information and the ORCID identification number(s) for the author(s) of this articlecan be found under: https://doi.org/10.

Pulsed electron-electron double resonance (PELDOR or DEER) spectroscopy is frequently used to determine distances between spin centres in biomacromolecular systems. Experiments where mutual orientations of the spin pair are selectively excited provide the so-called orientation-selective PELDOR data. This data is characterised by the orientation dependence of the modulation depth parameter and of the dipolar frequencies. This dependence has to be taken into account in the data analysis in order to extract distance distributions accurately from the experimental time traces. In this work, a fitting algorithm for such data analysis is discussed. The approach is tested on PELDOR data-sets from the literature and is compared with the previous results.

Site-directed spin labeling of RNA based on click chemistry is used in combination with pulsed electron-electron double resonance (PELDOR) to benchmark a nitroxide spin label, called here dŲ. We compare this approach with another established method that employs the rigid spin label Çm for RNA labeling. By using CD spectroscopy, thermal denaturation measurements, CW-EPR as well as PELDOR we analyzed and compared the influence of dŲ and Çm on a self-complementary RNA duplex. Our results demonstrate that the conformational diversity of dŲ is significantly reduced near the freezing temperature of a phosphate buffer, resulting in strongly orientation-selective PELDOR time traces of the dŲ-labeled RNA duplex.