Thesis Title: Subtitle - NMR Spectroscopy Research Group

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126 Chapter 4. Protein Structure Determination from Pseudocontact Shifts using ROSETTA. Table S4.2 Protein structures used to evaluate the performance of PCS-ROSETTA. Targets PCS-ROSETTA run a CS-ROSETTA run b rmsd c convergence d rmsd c convergence d protein G (A) 0.61 0.92 0.80 0.88 calbindin (B) 1.46 2.09 4.96 4.72 θ subunit (C) 1.30 0.55 1.56 2.25 ArgN e (D) 1.00 0.77 1.31 2.21 ArgN f (E) 0.83 0.94 1.65 5.43 N-calmodulin (F) 1.74 1.49 4.69 4.49 thioredoxin (G) 2.58 2.44 4.61 5.55 parvalbumin (H) 11.26 10.25 11.80 11.30 calmodulin (I) 2.80 2.12 6.35 2.94 ε186 g (J) 20.57 18.03 17.07 17.74 a The structures used to calculate the rmsds were identified using the combined PCS-score and ROSETTA full atom energy across only the core residues defined in SI Table S4.1. b The structures used to calculate the rmsds were identified by the ROSETTA full-atom energy across only the core residues defined in SI Table S4.1. c C α rmsd (with respect to the native structure) of the structure of lowest score, in Å. All C rmsd values were calculated using the core residues defined in SI Table S4.1. d Average C α rmsd calculated between the lowest score structure and the next four lowest scoring structure, in Å. The rmsd values were calculated using the core residues defined in SI Table S4.1. e PCSs measured with a covalent tag attached to the N-terminal domain of the E. coli arginine repressor (ArgN). f PCSs measured with a non-covalent tag bound to ArgN. g N-terminal 186 residues of the ε subunit of the E. coli polymerase III. Text S4.1 Fragment Assembly Using PCSs Only. In order to gain a better understanding of the merit of PCS data, we generated 10000 decoys per protein with all ROSETTA force field components turned off except for the PCS score. In seven of the ten protein structure calculations, the PCS score alone produced decoys with a C rmsd of less than 2.5 Å to the target structure (Figure S4.2, solid blue line). Control calculations without
4.8 Supporting information. 127 any scoring function produced not a single useful decoy. This highlights the power of PCS data to define the overall topology of a protein at the fragment assembly stage. The effect was particularly pronounced for the target proteins θ and ArgN (Figure S4.2 C and D). The second set of PCS data of ArgN (Table 4.1; structure E) yielded worse decoys in the PCS-only computations with PCS-ROSETTA than CS-ROSETTA. Remarkably, however, using the PCS score in combination with the ROSETTA force field yielded much better structures than when used separately (Figure S4.3 E). This shows that the PCS score adds information that is not captured by the ROSETTA energy score alone. Text S4.2 Scoring over Core Residues. Disordered residues can add noise to the ROSETTA energy, and this noise can prevent identification of low rmsd structures. Notably, three of the targets that succeeded under the PCS- ROSETTA protocol and failed under the CS-ROSETTA protocol have disordered termini accounting for ten or more residues each (Table S4.2-d: Targets C, D & E). In practice it is possible to experimentally determine the disordered character of a residue, so to compare the effect of disorder on the two protocols we produced an additional set of structures by removing disordered residues during the final rescoring step (cores defined in Table S4.1). When the core residues are perfectly defined, in this case by observation of the solved structures, the CS-ROSETTA protocol identifies low rmsd structures in four of the ten cases (including C, D & E), and shows convergence to a low rmsd structure in three of the ten cases [Table S4.2-c, d, CS-ROSETTA run]. In contrast, removing the disordered residues has little effect on PCS-ROSETTA’s rmsd values, suggesting that the combined PCS and ROSETTA score is less sensitive to disorder. The remaining targets had little disorder and removal of disordered terminal residues had little effect on the results.
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Computational study of proteins wit
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iii Schmitz C, Stanton-Cook MJ, Su
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v Acknowledgements I would like to
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vii Keywords paramagnetic nmr, pseu
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ix 2.3.3 Manual resonance assignmen
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xi List of Figures Figure 1.1 NMR e
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xv List of Abbreviations α Subunit
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Thesis Title: Subtitle - NMR Spectroscopy Research Group

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