Supplementary Text.

Here we introduce chiLife, a Python package for site-directed spin label (SDSL) modeling for electron paramagnetic resonance (EPR) spectroscopy, in particular double electron–electron resonance (DEER). It is based on in silico attachment of rotamer ensemble representations of spin labels to protein structures. chiLife enables the development of custom protein analysis and modeling pipelines using SDSL EPR experimental data. It allows the user to add custom spin labels, scoring functions and spin label modeling methods. chiLife is designed with integration into third-party software in mind, to take advantage of the diverse and rapidly expanding set of molecular modeling tools available with a Python interface. This article describes the main design principles of chiLife and presents a series of examples.</div

Comparison of distance distribution predictions using different scoring functions.

A) Definition and application of custom energy function using chiLife. B) Experimental distance distributions for two site pairs of MPB, taken from [59], are compared to the predicted distance distributions from spin labels modeled using a modified Lennard–Jones potential and a custom function that augments the same potential with an additional term to account for compensatory attractive forces with the solvent.</p

Comparison of a previously published ExoU–ubiquitin complex model and the best scoring model obtained by integrating chiLife and PyRosetta.

A) Docking funnel showing convergence towards the previously published complex using chiLife restraints. B) Cartoon structures of ExoU (gray) showing predicted locations of ubiquitin in the previously published model (purple) and in the PyRosetta model produced here (blue).</p

Illustration of chiLife’s SpinLabel object.

On the left, some of its user-accessible properties are shown. On the right, some useful methods are illustrated that allow users to modify or calculate new structures from the SpinLabel object.</p

Membrane docking of the cPLA2 C2 domain (PDBID 1BCI).

The cPLA2 C2 domain is shown as a gray cartoon. Spin centroids are shown as red spheres with their color saturation mapped to the experimental depth. Native side chains of spin labeled sites are shown as sticks. The blue grid indicates the phosphate plane of the model.</p

Local side chain repacking for R1 spin label ensembles at sites N124R1 and E281R1 on MBP (PDBID 1OMP).

A) Prediction of spin label ensembles without repacking. The protein structure is shown as a gray cartoon. Spin labels are shown as blue sticks. B) Energy trajectory of MCMC repacking, relative to the energy of the starting structure. C) Spin label (blue sticks) and neighboring side chain (green sticks) ensembles obtained from the repacking trajectory. D) Comparison of predicted distance distributions derived from the ensembles of the repacked and the original structures.</p

Three spin labels added to chiLife and attached to T4 lysozyme (PDBID: 2LZM) at site 109.

R3 (left, sticks with blue carbons) is a small highly mobile nitroxide label. Gd(III)-DO3A (center, sticks with dark red carbons) is a gadolinium-based spin label resistant to reduction. NOBA (right, sticks with green carbons) is a biorthogonal nitroxide. The surface is made by pseudo-atoms at the rotamer spin centers.</p

Illustration of how changes in backbone do not necessarily cause changes in distance distributions.

Top: Comparison of apo (blue, PDBID 1OMP) and holo (red, PDBID 1ANF) MBP structures and the locations of the model R1 spin labels (sticks) for the site pairs E38R1 S352R1 (A) and E45R1 S211R1 (B). Bottom: Comparison of apo and holo distance distributions for the two site pairs that both show significant changes in Cβ–Cβ backbone distance, indicated by small triangles at the base of the plots. The E38R1 S152R1 site pair on the left (C) shows a clear difference in the predicted distributions while the E45R1 S211R1 site pair on the right (D) shows very little change.</p

Spin labeling proteins and predicting spin label distance distributions.

A) chiLife script. B) Cartoon model of maltose binding protein (PDBID 1OMP) labeled with R1 at sites 238 and 275, showing the spin label ensembles (sticks) and weighted kernel density estimates of the spin centers (semitransparent surfaces). C) Comparison of the predicted distance distributions with the experimental distance distribution.</p

Structure and Dynamics of Type III Secretion Effector Protein ExoU As determined by SDSL-EPR Spectroscopy in Conjunction with De Novo Protein Folding

ExoU is a 74 kDa cytotoxin that undergoes substantial conformational changes as part of its function, that is, it has multiple thermodynamically stable conformations that interchange depending on its environment. Such flexible proteins pose unique challenges to structural biology: (1) not only is it often difficult to determine structures by X-ray crystallography for all biologically relevant conformations because of the flat energy landscape (2) but also experimental conditions can easily perturb the biologically relevant conformation. The first challenge can be overcome by applying orthogonal structural biology techniques that are capable of observing alternative, biologically relevant conformations. The second challenge can be addressed by determining the structure in the same biological state with two independent techniques under different experimental conditions. If both techniques converge to the same structural model, the confidence that an unperturbed biologically relevant conformation is observed increases. To this end, we determine the structure of the C-terminal domain of the effector protein, ExoU, from data obtained by electron paramagnetic resonance spectroscopy in conjunction with site-directed spin labeling and in silico de novo structure determination. Our protocol encompasses a multimodule approach, consisting of low-resolution topology sampling, clustering, and high-resolution refinement. The resulting model was compared with an ExoU model in complex with its chaperone SpcU obtained previously by X-ray crystallography. The two models converged to a minimal RMSD100 of 3.2 Å, providing evidence that the unbound structure of ExoU matches the fold observed in complex with SpcU