Fig. 2.
MTSL spin label observed in the Spa15 crystal structure. (a) Ribbon diagram of spin-labeled Spa15 dimer crystal structure, showing the two Spa15 subunits coloured blue to green from the N to C terminus, respectively. The MTSL spin label is atom coloured, highlighting the S–S bond, with the blue nitrogen bound to purple oxygen. The distance between the two spin labels is shown. (b) The spin label of Cys19 was highly ordered in the electron density at 1σ background. The label is shown to have only one conformation, from the MTSL O to ND1, due to particular stabilization by the MTSL C6 to CD1 of Ile17 and due to the steric restraints of the pocket. (c) The hydrophobic pocket within which the spin label (red) resides. Neighbouring Leu14, Ile17, Ile23, Ile35, Leu37, Ile43, Leu119, His120, and Tyr123 are shown. There is little difference between backbone structure for unlabeled (PDB 1RY9) (light blue) and labeled (colour scheme as for a) (dark blue-green) structures, with perhaps a subtle closing around the label in the latter. Side chains are similarly unaffected by the spin label, an exception being Ile17, which is placed further into the hydrophobic pocket in 1RY9 than in the labeled 2XGA structure. (d) Experimentally observed MTSL conformation (stick) compared to MMM calculated conformations. The most probable at 175 K (orange line; P = 0.17) is unlike the experimentally observed conformation and points away from the hydrophobic pocket. The most probable conformation at 50 K produced a DEER distance similar to that experimentally observed. However, comparison of this conformation (P = 0.65) with the experimental conformation shows that this is not due to the correct prediction of the experimental conformation.