Thesis Title: Subtitle - NMR Spectroscopy Research Group

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86 Chapter 3. Numbat: new user-friendly method built for automatic Δχ-tensor determination. selection of subsets of data. Numbat calls gnuplot to display the results in a Sanson- Flamsteed projection plot. 3.6.12 Output The list of PCS can be saved in Xplor-NIH format and in a Numbat-specific format. The weak molecular alignment in the magnetic field resulting from a non-vanishing Δχ-tensor can be described by an alignment tensor with principal axes parallel to those of the Δχ-tensor and axial and rhombic components that are directly proportional to Δχax and Δχrh, respectively (Tolman et al., 1995). Numbat calculates the RDC between two spins A and B for the situation of a completely rigid molecule, using (Bertini et al., 2002) (3.5) where γA and γB are the magnetogyric ratios of spins A and B, respectively, ħ the Planck constant divided by 2π, S the order parameter, rAB the internuclear distance, and AB, AB, AB the coordinates of the vector AB expressed in the Δχ-tensor frame. The RDC values are reported in Xplor-NIH (Schwieters et al., 2003, Schwieters et al., 2006) and Pales (Zweckstetter et al., 2000) format. Finally, Numbat can generate PDB files where the Δχ-tensor is reported in a format ready for use with MOLMOL or PyMOL for rigid-body docking alignment, or for further refinement by Xplor-NIH. 3.7 Study case The proteins ε and θ are subunits of the complex of proteins constituting E. coli DNA polymerase III. The complex between the N-terminal domain of ε (ε186) and θ has been extensively studied using PCS data (Pintacuda et al., 2006, Pintacuda et al., 2007). In light of the recent crystal structure of the complex between ε186 and the θ homolog HOT (Kirby et al., 2006), we illustrate in the following the features of Numbat by revisiting the <strong>NMR</strong> structure of the complex between ε186 and θ which was derived from PCS induced by Dy 3+ and Er 3+ ions bound to the natural metal-binding site of ε186 (Pintacuda et al., 2006).
3.7 Study case. 87 The coordinates of the A chain in the PDB deposition 2IDO (Kirby et al., 2006) was used as the structural model for ε186. The structural model of θ was conformer 10 of the <strong>NMR</strong> structure of θ in complex with ε186 (PDB accession code 2AXD; (Keniry et al., 2006)). This conformer was chosen because it has the lowest backbone RMSD to the HOT protein (2.1 Å) for residues 9-66 (the structurally defined region for which meaningful PCS could be measured). The experimentally determined PCS values of ε186 have been reported previously (Schmitz et al., 2006) and the PCS values of θ are provided in the Supporting Information. All Δχ-tensor optimizations were performed using Numbat including the RACS correction term and a tolerance value toli of zero for all spins. 3.7.1 Subunit ε186 Table 3.1 presents the results of the Δχ-tensor fit to the PCS measured for ε186. Initially, individual eight-variable Δχ-tensor optimizations were performed using the PCS data of each lanthanide (Table 3.1, columns 1 and 2). Next, the Numbat GUI was updated to display the deviations between the experimental and back-calculated PCS for the Δχ-tensors found. Several atoms showed deviations > 0.15 ppm between the experimental and back-calculated PCS (15 out of 199 and 8 out of 255 atoms in the case of Dy 3+ and Er 3+ , respectively. Without the RACS correction, deviations > 0.15 ppm where observed for 36 and 7 atoms, respectively). Assuming that these outliers were due to problematic measurements or inaccuracies of the 3D structure, these PCS were removed interactively using the GUI. Re-calculation of the Δχ-tensor was found not to change the fitted Δχ-tensor parameters significantly for any of the lanthanide ions (results not shown). This can be explained by the high quality and large number of experimental PCS data available for each lanthanide (backbone 13 C’, 15 N and 1 H N spins), resulting in robust fits of the Δχ-tensors. Table 3.1 Δχ-tensors determined by Numbat in the frames of the ε186 and θ molecule ε186 a θ b Individual c Combined d Individual c Combined d Fixed e Dy 3+ Er 3+ Dy 3+ Er 3+ Dy 3+ Er 3+ Dy 3+ Er 3+ Dy 3+ Er 3+ Δχax f 42.3 -10.6 42.3 -10.7 40.1 -13.0 40.2 -10.0 42.3 -10.7 Δχrh f 5.3 -5.1 5.3 -5.1 14.8 -6.5 14.9 -4.8 5.3 -5.1 α g 169.5 144.2 169.5 143.9 27.7 23.7 27.7 19.9 42.2 34.9 β g 30.2 29.1 30.2 29.2 114.6 108.8 113.9 118.2 119.2 121.5 γ g 134.6 126.9 134.7 126.8 28.4 170.6 27.3 177.7 44.7 177.4 mx h 29.4 29.3 29.4 29.4 6.2 9.5 6.4 6.4 4.3 4.3 my h 31.9 32.0 31.9 31.9 -7.5 -7.2 -7.5 -7.5 -5.5 -5.5
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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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xv List of Abbreviations α Subunit
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Thesis Title: Subtitle - NMR Spectroscopy Research Group

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