Benchmark Test and Guidelines for DEER/PELDOR Experiments on Nitroxide-Labeled Biomolecules

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.

Automated summary

Distance distribution information obtained by pulsed dipolar EPR spectroscopy plays a crucial role in structural biology research, providing unique constraints for integrative structural modeling. Quality standards for sample preparation, measurements, data conversion, interpretation, and reporting have been defined to ensure the reliability of structural models and biological conclusions, with support from a multi-laboratory benchmark study and analysis of data sets with known distance distribution ground truth. The guidelines mainly focus on proteins labeled with nitroxides and double electron-electron resonance measurements, while also offering suggestions for similar cases.