High-resolution crystal structure of spin labelled (T21R1) azurin from Pseudomonas aeruginosa: a challenging structural benchmark for in silico spin labelling algorithms

Abstract

BackgroundEPR-based distance measurements between spin labels in proteins have become a valuable tool in structural biology. The direct translation of the experimental distances into structural information is however often impaired by the intrinsic flexibility of the spin labelled side chains. Different algorithms exist that predict the approximate conformation of the spin label either by using pre-computed rotamer libraries of the labelled side chain (rotamer approach) or by simply determining its accessible volume (accessible volume approach). Surprisingly, comparisons with many experimental distances have shown that both approaches deliver the same distance prediction accuracy of about 3 Å.ResultsHere, instead of comparing predicted and experimental distances, we test the ability of both approaches to predict the actual conformations of spin labels found in a new high-resolution crystal structure of spin labelled azurin (T21R1). Inside the crystal, the label is found in two very different environments which serve as a challenging test for the in silico approaches.ConclusionsOur results illustrate why simple and more sophisticated programs lead to the same prediciton error. Thus, a more precise treatment of the complete environment of the label and also its interactions with the environment will be needed to increase the accuracy of in silico spin labelling algorithms.

EPR‐Based Approach for the Localization of Paramagnetic Metal Ions in Biomolecules

“…At the same time, the pronounced bimodal distance distribution found for mutant T21R 1 fits to the crystal structure of this mutant, revealing the presence of two MTSSL conformations (Figure 1 b). 15 Moreover, the Cu 2+ –MTSSL distances calculated for these conformations are in very good agreement with the distances determined by PELDOR (Table 1). In the three other mutants, the second distance peak appears only as a shoulder, but also for these cases the presence of two MTSSL conformations is the most likely explanation.…”

“… a) The structure of azurin (PDB 1E67) and the chosen mutation labeling sites (pink spheres). b) The crystal structure of the azurin mutant T21R 1 (PDB 4BWW) with the two different conformations of MTSSL overlaid 15. The distance vectors connecting the Cu 2+ ion with the oxygen atom of MTSSL in T21R 1 are shown by arrows.…”

PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution

“…Further, our structure is another indication that crystal structures of the R1 side chain can be good approximations for the rotameric state of the side chain in frozen solution (i.e., in PELDOR samples) (39,40).…”

“…The experimental and expected distributions for VcSiaP Q54R1/Q110R1 agree very well for both experiments (with and without Neu5Ac). A possible explanation for the shoulder at 30 Å is a second conformation of the R1 spin label, which has been frequently observed in available crystal structures of the R1 side chain (38)(39)(40).…”

EPR‐Based Approach for the Localization of Paramagnetic Metal Ions in Biomolecules

“…b) The crystal structure of the azurin mutant T21R 1 (PDB 4BWW) with the two different conformations of MTSSL overlaid. [15] The distance vectors connecting the Cu 2+ ion with the oxygen atom of MTSSL in T21R 1 are shown by arrows. The distances obtained by these three methods are shown in Figure 3 c and Table 1.…”