Benchmark Test and Guidelines for DEER/PELDOR Experiments on Nitroxide-Labeled Biomolecules
Olav Schiemann, Caspar A. Heubach, Dinar Abdullin, Katrin Ackermann, Mykhailo Azarkh, Elena G. Bagryanskaya, Malte Drescher, Burkhard Endeward, Jack H. Freed, Laura Galazzo, Daniella Goldfarb, Tobias Hett, Laura Esteban Hofer, Luis Fábregas Ibáñez, Eric J. Hustedt, Svetlana Kucher, Ilya Kuprov, Janet E. Lovett, Andreas Meyer, Sharon Ruthstein, Sunil Saxena, Stefan Stoll, Christiane R. Timmel, Marilena Di Valentin, Hassane S. Mchaourab, Thomas F. Prisner, Bela E. Bode, Enrica Bordignon, Marina Bennati, Gunnar Jeschke
Abstract
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.