Ural modeling of IPPmin based on SAXS data. A) Averaged molecular

Ural modeling of IPPmin based on SAXS data. A) Averaged molecular 15900046 envelope for IPPmin. The approximate envelope ?dimensions (in A) are illustrated. The two views are related by 90u rotation. B) The crystal structures of the individual subunits of the IPPmin complex, ILK-ARD/PINCH-1-LIM1 (PDB code: 3F6Q) and ILKpseudokinase (pKD)/a-parvin-CH2 (PDB code: 3KMU) used in rigid body modeling. ILK is colored MedChemExpress Eliglustat magenta, PINCH-1 is green, and a-parvin is blue. C) CORAL [36] rigid body model of IPPmin (ribbons, colored as in B) with the best statistical fit to the 50-14-6 experimental data (plotted in D). Overlaid is the averaged molecular envelope. 14 inter-domain dummy residues between the C-terminus of ILK-ARD and the N-terminus of ILKpKD, in the optimal conformation chosen by CORAL, are depicted as ?yellow spheres. The distance between the two subunits is 26 A. D) Fit of the theoretical scattering profile for the rigid body model (red line) with the experimental SAXS data (logarithmic). Residuals for the fit are shown below. doi:10.1371/journal.pone.0055591.gIPPmin adopts a predominantly compact conformation in solutionWe next assessed inter-domain flexibility in the IPPmin complex. A Porod-Debye plot of the IPPmin scattering data (Figure 4A) shows a plateau that fits the linear plot with a Porod Exponent of 4, consistent with a well-ordered, globular particle with little to no flexibility [33]. We also examined flexibility using the ensemble optimization method (EOM) [37]. The ILK-ARD/PINCH1LIM1 and ILK-pKD/a-parvin-CH2 structures were treated as rigid bodies connected by a flexible linker of 14 dummy residues missing from the crystal structures (residues 171?84; Figure 1A), and a pool of 10,000 individual models were generated containing a random sampling of linker conformations and subunit positions avoiding steric clashes to cover the range of configurational space.SAXS Analysis of the IPP ComplexFigure 4. Flexibility analysis of IPPmin. A) Porod-Debye plot of IPPmin SAXS data (open circles) shows a linear plateau (red line) consistent with a folded, globular protein with little flexibility. B thru D) Ensemble Optimization Method (EOM). B) Fits of the theoretical scattering profiles for the selected ensembles (containing 20 models: red line, 2 models: green line, 1 model: blue line) with the experimental SAXS data (logarithmic scale; top) ?and the residuals of the fits (bottom). C) Rg size distribution (A) for selected ensemble (20 models, red) compared with the pool of 10,000 models ?(grey) used for EOM showing two populations of IPPmin structures. The dashed line represents the average Rg value (34.7 A) of the predominant conformation in the optimized ensemble. D) EOM-generated model representing the most representative structure of IPP in the optimized ensemble (NSD = 1.3), which is overlaid with the averaged molecular envelope. The Dmax of the respective models is shown. Protein domains are labeled and colored as in Figure 3B. doi:10.1371/journal.pone.0055591.gA genetic algorithm was then employed to select an optimized ensemble of models whose combined theoretical scattering curve best represents the IPPmin experimental scattering profile. EOM analysis of our IPPmin SAXS data yields an optimized ensemble (containing 20 models) that fits the experimental scattering curve with x value of 1.5 (Figure 4B). An ensemble containing as few as 2 models fits the experimental data equally well as the larger 20 model ensemble (x = 1.5, Figure 4B), suggest.Ural modeling of IPPmin based on SAXS data. A) Averaged molecular 15900046 envelope for IPPmin. The approximate envelope ?dimensions (in A) are illustrated. The two views are related by 90u rotation. B) The crystal structures of the individual subunits of the IPPmin complex, ILK-ARD/PINCH-1-LIM1 (PDB code: 3F6Q) and ILKpseudokinase (pKD)/a-parvin-CH2 (PDB code: 3KMU) used in rigid body modeling. ILK is colored magenta, PINCH-1 is green, and a-parvin is blue. C) CORAL [36] rigid body model of IPPmin (ribbons, colored as in B) with the best statistical fit to the experimental data (plotted in D). Overlaid is the averaged molecular envelope. 14 inter-domain dummy residues between the C-terminus of ILK-ARD and the N-terminus of ILKpKD, in the optimal conformation chosen by CORAL, are depicted as ?yellow spheres. The distance between the two subunits is 26 A. D) Fit of the theoretical scattering profile for the rigid body model (red line) with the experimental SAXS data (logarithmic). Residuals for the fit are shown below. doi:10.1371/journal.pone.0055591.gIPPmin adopts a predominantly compact conformation in solutionWe next assessed inter-domain flexibility in the IPPmin complex. A Porod-Debye plot of the IPPmin scattering data (Figure 4A) shows a plateau that fits the linear plot with a Porod Exponent of 4, consistent with a well-ordered, globular particle with little to no flexibility [33]. We also examined flexibility using the ensemble optimization method (EOM) [37]. The ILK-ARD/PINCH1LIM1 and ILK-pKD/a-parvin-CH2 structures were treated as rigid bodies connected by a flexible linker of 14 dummy residues missing from the crystal structures (residues 171?84; Figure 1A), and a pool of 10,000 individual models were generated containing a random sampling of linker conformations and subunit positions avoiding steric clashes to cover the range of configurational space.SAXS Analysis of the IPP ComplexFigure 4. Flexibility analysis of IPPmin. A) Porod-Debye plot of IPPmin SAXS data (open circles) shows a linear plateau (red line) consistent with a folded, globular protein with little flexibility. B thru D) Ensemble Optimization Method (EOM). B) Fits of the theoretical scattering profiles for the selected ensembles (containing 20 models: red line, 2 models: green line, 1 model: blue line) with the experimental SAXS data (logarithmic scale; top) ?and the residuals of the fits (bottom). C) Rg size distribution (A) for selected ensemble (20 models, red) compared with the pool of 10,000 models ?(grey) used for EOM showing two populations of IPPmin structures. The dashed line represents the average Rg value (34.7 A) of the predominant conformation in the optimized ensemble. D) EOM-generated model representing the most representative structure of IPP in the optimized ensemble (NSD = 1.3), which is overlaid with the averaged molecular envelope. The Dmax of the respective models is shown. Protein domains are labeled and colored as in Figure 3B. doi:10.1371/journal.pone.0055591.gA genetic algorithm was then employed to select an optimized ensemble of models whose combined theoretical scattering curve best represents the IPPmin experimental scattering profile. EOM analysis of our IPPmin SAXS data yields an optimized ensemble (containing 20 models) that fits the experimental scattering curve with x value of 1.5 (Figure 4B). An ensemble containing as few as 2 models fits the experimental data equally well as the larger 20 model ensemble (x = 1.5, Figure 4B), suggest.