Cross-validation of distance measurements in proteins

August 02, 2022

PD Dr. Gregor Hagelueken (left) and Dr. Martin Peter (right) (picture: Johann Saba / University Hospital Bonn)


Large scale analysis of PELDOR/DEER and smFRET as complementary tools in structural biology

In order to investigate intermolecular distances, conformational changes or structural heterogeneity of biological molecules, two different methods are widely used: Pulsed electron-electron double resonance spectroscopy (PELDOR/DEER) and single-molecule Förster resonance energy transfer spectroscopy (smFRET). Scientists of the excellence cluster ImmunoSensation2 at the University of Bonn and of the Ludwig-Maximilians-Universität (LMU) Munich have now compared the accuracy of the two methods. The study has now been published in the journal Nature Communications.

The researchers from PD Dr. Gregor Hagelueken's group at the Institute of Structural Biology of the University Hospital Bonn (UKB) used four different proteins as model structures to be analyzed: Three different substrate binding proteins (SBPs), and an actin-binging virulence factor, all four exhibiting large conformational changes upon ligand binding. PELDOR spectroscopy was used to determine inter-probe distances at the nanometre scale and detect conformational changes of the macromolecules in their (frozen) solution state. Identical experiments were carried out at the research group of Prof. Dr. Thorben Cordes at LMU Munich, using smFRET spectroscopy.

"Although both methods are used very frequently, no one has yet systematically investigated whether the results are really comparable," says Hagelueken. Although it turned out that the results were comparable in most cases, the researchers encountered inconsistencies in two cases. Bonn post-doctoral researcher Martin Peter says, "We then thoroughly investigated what caused the differences and found what we were looking for. In one case, it turned out that the dye molecules stuck to the protein and thus falsified the measurement." In the second case, the addition of a type of antifreeze, which was necessary because of the low measurement temperature of below -220 degrees Celsius, led to unexpected deviations. "We were able to show that despite the high accuracy of the methods, re-measuring with another nano ruler is always a good idea," Hagelueken says.


Publication: Martin F. Peter, Christian Gebhardt, Rebecca Mächtel, Gabriel G. Moya Muñoz Janin Glaenzer, Alessandra Narducci, Gavin H. Thomas, Thorben Cordes, Gregor Hagelueken: Cross-validation of distance measurements in proteins by PELDOR/DEER and single-molecule FRET; Nature Communications;



PD Dr. Gregor Hagelueken
Institute for Structural Biology at the University Hospital Bonn
Phone: +49 228 287-51200
Email: hagelueken(at)