Thesis Title: Subtitle - NMR Spectroscopy Research Group

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90 Chapter 3. Numbat: new user-friendly method built for automatic Δχ-tensor determination. Er 3+ ). Like for ε186, removal of these PCS from the optimization did not significantly change the parameters of the fitted Δχ-tensors. While the Δχax and Δχrh values of Er 3+ determined from the PCS observed for θ and 186 were very similar, the Δχrh value of the Dy 3+ tensor found for θ was almost three times larger than that found for ε186 6 . We subsequently performed an error analysis for θ as for the ε186 subunit, introducing either random variations into the atomic positions of θ according to a Gaussian distribution with a standard deviation ζ of 0.5 Å or using a random selection of only 80% of the measured PCS. In either case, the Δχ-tensor parameters of θ proved to be less well defined than those of ε186 (Table 3.2) and Figure 1.14.b. As θ samples a relatively small and remote volume of the Δχ-tensors due to its spatial separation from the metal ion, one would expect a less accurate determination of the Δχ-tensors from the θ data. The effect could be exacerbated by inaccuracies of the <strong>NMR</strong> structure. In order to compensate for the smaller number of experimentally determined PCS available for θ (only 1 H N spins) and the poorer quality of the Δχ-tensors fitted, we performed another fit with Δχax and Δχrh fixed to the values determined for ε186 (Table 3.1, columns 9 and 10). Analysis of the experimental versus back-calculated PCS, both for the eight- and six-variable fits of the Δχ- tensor to θ, showed that the PCS deviations were similar in magnitude and trends. Therefore, constraining Δχax and Δχrh did not significantly deteriorate the quality of the fit, despite considerable changes of the Δχ-tensor parameters (Table 3.1). The variability of the Δχ-tensor parameters over all the 12 deposited θ conformers in 2AXD using the fixed optimisation scheme is provided in the Supporting Information. 3.7.3 Modelling the complex between ε186 and θ Numbat facilitates the modelling of protein-protein complexes by listing coordinates of the Δχ-tensor axes together with the protein coordinates in files in PDB format. Superimposition of the Δχ-tensors fitted to ε186 and θ for each lanthanide ion yields the three-dimensional structure of the ε186/θ complex by straightforward rigid-body docking. Standard PyMOL or MOLMOL commands 6 The discrepancy of the Rhombic component would not necessarily affect the rigid body docking of the complex, as only the orientation of the Δχ-tensors and the coordinates of the paramagnetic center are used.
3.7 Study case. 91 can be used to align the Δχ-tensors. Numbat reports the coordinate system of the Δχ-tensor in such a way that all four degenerate solutions arising from the symmetry of the Δχ-tensor about the x, y and z axes (Figure 3.4) can easily be visualised. Identification of the correct solution requires additional information, such as proper steric interactions, chemical shift perturbation data or knowledge of the biological function of the complex. The most objective way, however, is by simultaneous evaluation of the Δχ-tensors of different lanthanides (Pintacuda et al., 2006). In the case of the complex between ε186 and θ, the Δχ-tensor frames of Dy 3+ and Er 3+ share a common origin for both proteins. Seven coordinates are necessary to define two Δχ-tensor frames sharing the same origin. Because of the second Δχ-tensor, the degeneracy of Figure 3.4 is broken. There are exactly 16 possibilities to align two pairs of Δχ-tensor. The lowest RMSD value resulting from all 16 possible 7-coordinate alignments between the two combined Δχ-tensors identified a single relative orientation of the two proteins as the best solution. The position of θ relative to ε186 derived from PCS data in this way was also the correct solution. It agreed with a model of the complex obtained by superimposition of θ onto HOT in the ε186/HOT complex, with a backbone RMSD of 4.4 Å. Similarly for the Δχ-tensor of θ calculated with fixed Δχax and Δχrh values, a backbone RMSD of 4.3 Å was calculated relative to HOT. When PCS data from only Dy 3+ or Er 3+ were used, the backbone RMSD values were, respectively, 4.2 Å and 4.4 Å for the best fit to the ε186/HOT complex. The model of the ε186/θ complex derived from the fixed, Dy 3+ and Er 3+ data sets is displayed in Figure 3.5.
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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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xiii
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xv List of Abbreviations α Subunit
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2 Chapter 1. Introduction. The scal
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Page 109 and 110: 3.9 Acknowledgment. 93 variables, t
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Thesis Title: Subtitle - NMR Spectroscopy Research Group

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