Background

Site-directed spin labeling (SDSL) is a common technique to investigate structure and dynamics of macromolecular systems. Covalentry attached spin labels are introduced to the system and induce electron spin resonance.

Double electron electron spin resonance (DEER) is an EPR technique for measuring distances between two spin labels that have been covalently attached to a protein. Two cysteine residues are introduced into the protein and subsequently labelled.

Paramagnetic relaxation enhancement (PRE) is an NMR technique for measuring distances between a spin label and the amide protons of the protein backbone. One cysteine residue is introduced at the position of the label.

The positions are chosen to report on the expected conformational change. A commonly used spin label is (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)-methanethiosulfonate (MTSL). MTSL has a linker with five rotatable bonds and is therefore very flexible.

Method

The distance distributions between the two spin labels (DEER) or one spin label and the amide protons are measured by experiments (PRE) are typically broad and often multi-modal. The distributions are therefore a convolution of the flexibility of the MTSL spin label and the conformational spread of the proteins in the sample. In order to compare a structural ensemble (e.g., from MD simulations or modelling) to experimental DEER or PRE data, we developed a post-processing method that

  1. maps rotamer libraries of the MTSL spin label onto each position,

  2. discards those rotamers that sterically clash with the protein (typically distances <2 Å) and

  3. calculates all (weighted) distance pairs between the remaining rotamers and

  4. thereby estimates a distance distribution for that structure.

The code was written in Python using the MDAnalysis library [Michaud-Agrawal2011] [Gowers2016] and Rotamer libraries for MTSL (2011, 2015) [Polyhach2011].

Our approach improves upon the existing method [Polyhach2011] by increasing computational efficiency and implementing, via the MDAnalysis library, analysis of ensembles of hundreds of structures, which allowed us to estimate distance distributions for entire simulation trajectories. In the case of PRE measurements, it enables the user to calculate back the transverse relaxation enhancement to compare raw data without calculating the distances based on the experiment.

In the case of MTSL, the distances are determined by considering the position of the free electron located between nitrogen (N1) and (O1).

Example applications

Examples of the application of this approach for comparing experimental DEER data to molecular dynamics (MD) simulations can be found in [Stelzl2014] and [Fowler2015].