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Structural and Chemical Biology @ Vanderbilt University |
De novo High-Resolution Protein Structure Elucidation from
sparse EPR Data
Members: Nathan Alexander, Stephanie Hirst
all research projects
Figure 1
Spin label structure and dynamics determined by Rosetta rotamer libraries
The major limitation inherent to electron paramagnetic resonance (EPR) is that the exact position of the spin label that projects from the protein backbone is unknown. This limits the amount of detail that can be extracted from the EPR experiment, especially in the case of distance measurements. In order to overcome this drawback, we introduce a rotamer library of the methanethiosulfonate spin label (MTSSL) into the protein modeling program Rosetta. Spin label rotamers have been derived from conformations observed in crystal structures of spin labeled T4-lysozyme. The crystal structures reveal preferred combination of χ1 and χ2 angles. As χ3, χ4, and χ5 are often not observable in the crystal structures, the rotamer set enumerates preferred combinations of these angles from experiment and computation. Rotamers which have clashes are removed from the library, and spin label conformations are systematically modeled and evaluated using the Rosetta Monte Carlo energy function. The method was benchmarked using structurally determined single MTSSL mutants of T4-lysozyme and double mutants for which EPR distances were measured. The results indicate that Rosetta is able to predict important aspects of the spin label’s conformation. In particular, an accurate reproduction of experimental distances and distance distributions observed for many of the T4-lysozyme double mutants is found.
The mean (left) and standard deviation (right) of distances obtained from
the rotamer library compared to the experimentally measured mean and
standard deviation EPR distances for T4-lysozyme. (left) The mean distance
for the rotamer library (y-axis) is calculated for each double mutant
using the most energetically favorable models after all pair wise
combinations are systematically sampled. The distance is calculated
between the oxygen of the nitroxide on the two spin labels. The x-axis
gives the experimentally determined mean distance of the two spin labels
for the corresponding double mutant. The line is fit to the data and is
approximately y=x. (right) Same as above except with the standard
deviation of the spin label distances calculated rather than the mean
distance.
Members: Nathan Alexander, Stephanie Hirst
all research projects
Figure 1
Spin label structure and dynamics determined by Rosetta rotamer libraries
The major limitation inherent to electron paramagnetic resonance (EPR) is that the exact position of the spin label that projects from the protein backbone is unknown. This limits the amount of detail that can be extracted from the EPR experiment, especially in the case of distance measurements. In order to overcome this drawback, we introduce a rotamer library of the methanethiosulfonate spin label (MTSSL) into the protein modeling program Rosetta. Spin label rotamers have been derived from conformations observed in crystal structures of spin labeled T4-lysozyme. The crystal structures reveal preferred combination of χ1 and χ2 angles. As χ3, χ4, and χ5 are often not observable in the crystal structures, the rotamer set enumerates preferred combinations of these angles from experiment and computation. Rotamers which have clashes are removed from the library, and spin label conformations are systematically modeled and evaluated using the Rosetta Monte Carlo energy function. The method was benchmarked using structurally determined single MTSSL mutants of T4-lysozyme and double mutants for which EPR distances were measured. The results indicate that Rosetta is able to predict important aspects of the spin label’s conformation. In particular, an accurate reproduction of experimental distances and distance distributions observed for many of the T4-lysozyme double mutants is found.