Ing limited flexibility. A single model ensemble, in contrast, fits the

Ing limited flexibility. A single model ensemble, in contrast, fits the data slightly less well, with x = 1.8 (momelotinib chemical information Figure 4B), suggesting the potential for a small population of a second conformation of IPPmin. However, since the fit to the experimental data of the rigid body model (x = 1.4, Figure 3) is as good as the EOM optimized ensemble (x = 1.5), our data support a structure in which IPPmin exhibits limited flexibility. Comparing the Rg distributions of the optimized ensemble with the random pool, we find that the ensemble displays a more ?narrowed Rg distribution, with a major peak at Rg = 34.7 A (Figure 4C). This is in good agreement with the value calculated from the scattering curve (Table 1), and represents a predominant, compact IPPmin conformation in solution. We also observe a ?second minor, broad Rg peak above 40 A, which may indicate the presence of a small fraction of more extended IPPmin particles in ?solution (models with Rg above 40 A are selected at a frequency of 10 in the optimized ensemble). When repeating EOM analysis with SAXS data collected on lower IPPmin concentrations, we find that the trend in Rg distributions is largely unaffected by concentration (not shown) suggesting that the more elongated particle does not represent a concentration-dependent aggregate of IPPmin. However, we cannot exclude the possibility that the small peak at higher Rg MedChemExpress PF-00299804 values is an artifact of modeling and/or over-fitting of the high angle scattering data, or that a smallpercentage of IPPmin forms aggregates in all concentrations measured. We next assessed the structural variability in the selected ensemble by superposition using normalized spatial discrepancy (NSD) values [38]. The optimized ensemble has a NSD value of 1.460.1, lower than the NSD value for a set of 100 randomlychosen conformers from the pool (NSD = 1.660.2), consistent with a predominant IPPmin particle in solution. The most representative model from the optimized ensemble, which shows the smallest average variation (NSD = 1.3), adopts a somewhat compact shape that fits well with the molecular envelope ?(Figure 4D). This model has a Rg value of 35.4 A and Dmax of ?128.7 A, consistent with values calculated from the scattering curve (Table 1). Taken together, the results from EOM analysis support that IPPmin adopts a predominantly compact structure in solution with limited flexibility.ILK contains an unstructured inter-domain linkerThe N-terminal ILK-ARD and C-terminal ILK-pKD subunits are separated by a 14-residue linker (Figure 1A) that sequence profile analysis suggests is unstructured/disordered (PSIpred [41], DISOPRED [42], PrDOS [43], DisEMBL [44], data not shown). From EOM analysis, the predominant IPPmin structure is somewhat compact (Figure 3B and 4C), with Dmax values consistent with an average inter-subunit linker of approximately ?25 A. Similarly, rigid body modeling results in a linker of ?approximately 19 A. Considering that a fully extended linker ?could be as long as 50 A, this shorter average distance raises the possibility that the linker contains secondary structure and/or is partially structured through interactions with either the N-terminal ARD or C-terminal pKD of ILK. We therefore probed disorder inSAXS Analysis of the IPP ComplexFigure 5. An unstructured linker in ILK connects the N- and C-terminal subunits of IPP. A) Limited trypsin proteolysis of purified IPPmin complex (lanes 2 through 6) supports that the linker in ILK is unstructured. The.Ing limited flexibility. A single model ensemble, in contrast, fits the data slightly less well, with x = 1.8 (Figure 4B), suggesting the potential for a small population of a second conformation of IPPmin. However, since the fit to the experimental data of the rigid body model (x = 1.4, Figure 3) is as good as the EOM optimized ensemble (x = 1.5), our data support a structure in which IPPmin exhibits limited flexibility. Comparing the Rg distributions of the optimized ensemble with the random pool, we find that the ensemble displays a more ?narrowed Rg distribution, with a major peak at Rg = 34.7 A (Figure 4C). This is in good agreement with the value calculated from the scattering curve (Table 1), and represents a predominant, compact IPPmin conformation in solution. We also observe a ?second minor, broad Rg peak above 40 A, which may indicate the presence of a small fraction of more extended IPPmin particles in ?solution (models with Rg above 40 A are selected at a frequency of 10 in the optimized ensemble). When repeating EOM analysis with SAXS data collected on lower IPPmin concentrations, we find that the trend in Rg distributions is largely unaffected by concentration (not shown) suggesting that the more elongated particle does not represent a concentration-dependent aggregate of IPPmin. However, we cannot exclude the possibility that the small peak at higher Rg values is an artifact of modeling and/or over-fitting of the high angle scattering data, or that a smallpercentage of IPPmin forms aggregates in all concentrations measured. We next assessed the structural variability in the selected ensemble by superposition using normalized spatial discrepancy (NSD) values [38]. The optimized ensemble has a NSD value of 1.460.1, lower than the NSD value for a set of 100 randomlychosen conformers from the pool (NSD = 1.660.2), consistent with a predominant IPPmin particle in solution. The most representative model from the optimized ensemble, which shows the smallest average variation (NSD = 1.3), adopts a somewhat compact shape that fits well with the molecular envelope ?(Figure 4D). This model has a Rg value of 35.4 A and Dmax of ?128.7 A, consistent with values calculated from the scattering curve (Table 1). Taken together, the results from EOM analysis support that IPPmin adopts a predominantly compact structure in solution with limited flexibility.ILK contains an unstructured inter-domain linkerThe N-terminal ILK-ARD and C-terminal ILK-pKD subunits are separated by a 14-residue linker (Figure 1A) that sequence profile analysis suggests is unstructured/disordered (PSIpred [41], DISOPRED [42], PrDOS [43], DisEMBL [44], data not shown). From EOM analysis, the predominant IPPmin structure is somewhat compact (Figure 3B and 4C), with Dmax values consistent with an average inter-subunit linker of approximately ?25 A. Similarly, rigid body modeling results in a linker of ?approximately 19 A. Considering that a fully extended linker ?could be as long as 50 A, this shorter average distance raises the possibility that the linker contains secondary structure and/or is partially structured through interactions with either the N-terminal ARD or C-terminal pKD of ILK. We therefore probed disorder inSAXS Analysis of the IPP ComplexFigure 5. An unstructured linker in ILK connects the N- and C-terminal subunits of IPP. A) Limited trypsin proteolysis of purified IPPmin complex (lanes 2 through 6) supports that the linker in ILK is unstructured. The.