E 1). Next, the molecular weights based on Porod volumes and forward scattering were computed and compared with the expected molecular weight of the IPPmin complex (Table 1). Taken together with the elution profile from sizeexclusion chromatography (Figure 1E), we conclude that the IPPmin protein complex is monodisperse and monomeric in solution. The pairwise distribution functions, P(R), which reflects the inter-atomic distance distributions, were then determined, and are very similarly shaped for each of the IPPmin concentrations (Figure 2C). The asymmetric P(R) functions, which tail-off at higher q values, are consistent with a slightly elongated molecule ?with an asymmetric shape. The P(R) functions peak around 35 A ?, potentially indicating 
a second with a small shoulder around 50 A structural unit. Dimensionless Kratky plots show the characteristic globular peak for a folded protein (Figure 2D). Since no aggregation or repulsion is evident in the samples and since consistent Rg, Dmax and molecular weight values (Table 1) indicate no conformational Autophagy change with concentration, `zero extrapolation’ was not performed and data from the highest concentration of IPPmin (7.0 mg/ml) was used 
for all subsequent analysis.SAXS-based structural modeling of IPPWe performed structural modeling of the SAXS data using two different approaches. Using the P(R) function, ten individual ab initio molecular envelopes (dummy beads models) were reconstructed and averaged. The averaged envelope reveals a slightly extended shape that resembles a bicorne hat (Figure 3A) with ?dimensions 120660640 A, consistent with the experimentally determined Rg and Dmax values (Table 1). The envelope is asymmetric on its long axis, with one end slightly larger than the other. We next conducted rigid body modeling of the two subunits of IPPmin with CORAL [36]. Based on the protein boundaries in the available crystal structures versus our full-length ILK construct, the un-modeled linker between the ILK-ARD and ILK-pKD subunits is 14 residues (residues 171?84; Figure 1A). In rigid body analysis, the relative orientation between ILK-ARD/ PINCH1-LIM1 and ILK-pKD/a-parvin-CH2 (Figure 3B) was refined by simulated annealing using a Autophagy pre-calculated library of random, self-avoiding loops containing 14 dummy residues to constrain the distance between the two subunits, in order to best fit the experimental scattering data. This results in a model of IPPmin with overall shape similar 1662274 to the averaged molecular envelope, ?with an inter-domain distance of approximately 26 A (Figure 3C). The rigid body model fits well with the experimental data, with a x value of 1.4 (Figure 3D).Figure 3. Structural modeling of IPPmin based on SAXS data. A) Averaged molecular 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.E 1). Next, the molecular weights based on Porod volumes and forward scattering were computed and compared with the expected molecular weight of the IPPmin complex (Table 1). Taken together with the elution profile from sizeexclusion chromatography (Figure 1E), we conclude that the IPPmin protein complex is monodisperse and monomeric in solution. The pairwise distribution functions, P(R), which reflects the inter-atomic distance distributions, were then determined, and are very similarly shaped for each of the IPPmin concentrations (Figure 2C). The asymmetric P(R) functions, which tail-off at higher q values, are consistent with a slightly elongated molecule ?with an asymmetric shape. The P(R) functions peak around 35 A ?, potentially indicating a second with a small shoulder around 50 A structural unit. Dimensionless Kratky plots show the characteristic globular peak for a folded protein (Figure 2D). Since no aggregation or repulsion is evident in the samples and since consistent Rg, Dmax and molecular weight values (Table 1) indicate no conformational change with concentration, `zero extrapolation’ was not performed and data from the highest concentration of IPPmin (7.0 mg/ml) was used for all subsequent analysis.SAXS-based structural modeling of IPPWe performed structural modeling of the SAXS data using two different approaches. Using the P(R) function, ten individual ab initio molecular envelopes (dummy beads models) were reconstructed and averaged. The averaged envelope reveals a slightly extended shape that resembles a bicorne hat (Figure 3A) with ?dimensions 120660640 A, consistent with the experimentally determined Rg and Dmax values (Table 1). The envelope is asymmetric on its long axis, with one end slightly larger than the other. We next conducted rigid body modeling of the two subunits of IPPmin with CORAL [36]. Based on the protein boundaries in the available crystal structures versus our full-length ILK construct, the un-modeled linker between the ILK-ARD and ILK-pKD subunits is 14 residues (residues 171?84; Figure 1A). In rigid body analysis, the relative orientation between ILK-ARD/ PINCH1-LIM1 and ILK-pKD/a-parvin-CH2 (Figure 3B) was refined by simulated annealing using a pre-calculated library of random, self-avoiding loops containing 14 dummy residues to constrain the distance between the two subunits, in order to best fit the experimental scattering data. This results in a model of IPPmin with overall shape similar 1662274 to the averaged molecular envelope, ?with an inter-domain distance of approximately 26 A (Figure 3C). The rigid body model fits well with the experimental data, with a x value of 1.4 (Figure 3D).Figure 3. Structural modeling of IPPmin based on SAXS data. A) Averaged molecular 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.
