The IPP complex forms a stable, monodisperse, heterotrimeric complex.

<p><b>A</b>) Schematic diagram of the IPP complex: Integrin-linked kinase (ILK; magenta), PINCH (green) and Parvin (blue). ILK is the hub of the complex, and binds the LIM1 domain of PINCH-1 via its N-terminal ankyrin-repeat domain (ARD), and the C-terminal calponin homology (CH2) domain of α-parvin via its C-terminal pseudokinase domain (pKD) to form the IPP_min complex. The 14 residue inter-domain linker in ILK is shown. The lengths of the proteins are drawn approximately to scale. <b>B</b>) Co-expression of GST-ILK and (His)-α-parvin-CH2 in <i>E. coli</i>. Codon-optimized cDNA encoding full-length human ILK shows increased expression relative to the native ILK cDNA. (His)-PINCH-1-LIM is expressed alone in <i>E. coli</i>. <b>C</b>) TEV proteolysis removes the GST- and (His)-tags. <b>D</b>) Purified IPP_min complex is resolved by SDS-PAGE and stained with Coomassie blue (C.B.) to show a high level of purity. Anti-ILK immunoblot confirms the presence of ILK in the complex. <b>E</b>) Gel-filtration chromatography of IPP_min reveals a monodisperse protein species. The elution volume is consistent with a monomeric protein complex. The void volume is indicated. <b>F</b>) Native gel electrophoresis of purified IPP_min indicates that IPP is a stable protein complex. Purified IPP_min protein alone, and IPP_min plus added excess PINCH-1-LIM1 and/or α-parvin-CH2 proteins are resolved by native gel electrophoresis and visualized by Coomassie blue staining.</p

SAXS-derived size parameters for IPP_min.

a<p>Determined by Guinier approximation in Primus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055591#pone.0055591-Konarev1" target="_blank">[29]</a>.</p>b<p>Determined in AutoGNOM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055591#pone.0055591-Petoukhov1" target="_blank">[30]</a>.</p>c<p>Expected molecular weight = 73,625 Da.</p

Structural modeling of IPP_min based on SAXS data.

<p><b>A</b>) Averaged molecular envelope for IPP_min. The approximate envelope dimensions (in Å) are illustrated. The two views are related by 90° rotation. <b>B</b>) The crystal structures of the individual subunits of the IPP_min complex, ILK-ARD/PINCH-1-LIM1 (PDB code: 3F6Q) and ILK-pseudokinase (pKD)/α-parvin-CH2 (PDB code: 3KMU) used in rigid body modeling. ILK is colored magenta, PINCH-1 is green, and α-parvin is blue. <b>C</b>) CORAL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055591#pone.0055591-Petoukhov2" target="_blank">[36]</a> rigid body model of IPP_min (ribbons, colored as in <b>B</b>) with the best statistical fit to the experimental data (plotted in <b>D</b>). Overlaid is the averaged molecular envelope. 14 inter-domain dummy residues between the C-terminus of ILK-ARD and the N-terminus of ILK-pKD, in the optimal conformation chosen by CORAL, are depicted as yellow spheres. The distance between the two subunits is 26 Å. <b>D</b>) 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.</p

SAXS analysis for IPP_min reveals a globular heterotrimeric complex.

<p><b>A</b>) SAXS intensity profiles (logarithmic) for four concentrations of the IPP_min complex. <b>B</b>) Linearity of Guinier plots with manual selection of Guinier region. The <i>R</i>_g values are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055591#pone-0055591-t001" target="_blank"><b>Table 1</b></a>. Automatic Guinier analysis performed in AutoRG <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055591#pone.0055591-Konarev1" target="_blank">[29]</a>, which is consistent with the analysis shown here, is presented in the Supporting Information. <b>C</b>) Normalized pair distribution functions P(<i>R</i>) calculated automatically with AutoGNOM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055591#pone.0055591-Petoukhov1" target="_blank">[30]</a>. <b>D</b>) Dimensionless Kratky plots support a globular shape.</p

An unstructured linker in ILK connects the N- and C-terminal subunits of IPP.

<p><b>A</b>) Limited trypsin proteolysis of purified IPP_min complex (lanes 2 through 6) supports that the linker in ILK is unstructured. The N-terminal IPP subunit (ILK-ARD/PINCH-1-LIM1, lane 7) and α-parvin-CH2 alone (lane 8) are included for comparison. Molecular weight markers (in kDa) are shown. <b>B</b>) Gel-filtration chromatography of the full-length IPP_min protein (lane 1 from part D) and trypsin proteolyzed subunit fragments (lane 6 from part D) reveals no apparent interaction between the N- and C-terminal subunits of the IPP complex.</p

Flexibility analysis of IPP_min.

<p><b>A</b>) Porod-Debye plot of IPP_min SAXS data (open circles) shows a linear plateau (red line) consistent with a folded, globular protein with little flexibility. <b>B thru D</b>) Ensemble Optimization Method (EOM). <b>B</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). <b>C</b>) <i>R</i>_g size distribution (Å) for selected ensemble (20 models, red) compared with the pool of 10,000 models (grey) used for EOM showing two populations of IPP_min structures. The dashed line represents the average <i>R</i>_g value (34.7 Å) of the predominant conformation in the optimized ensemble. <b>D</b>) 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 D_max of the respective models is shown. Protein domains are labeled and colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055591#pone-0055591-g003" target="_blank">Figure 3B</a>.</p

Cocktails that produced visually recognizable crystals in the clusters identified in Figure 7.

<p>Cocktails that produced visually recognizable crystals in the clusters identified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100782#pone-0100782-g007" target="_blank">Figure 7</a>.</p

Regions of crystallization space where hits for BfR192 were found.

<p>Out of the 28 clusters, 11 were identified containing at least 1 crystal hit. The full list is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100782#pone-0100782-t004" target="_blank">Table 4</a>.</p

Each of these heatmaps represents a metric comparison between two consecutive generations of a screen from the Hauptman-Woodward Medical Research Institute [8].

<p>The screens have 1,536 cocktails, and the heatmaps can be viewed as overlays of the 1,536-well plate in which these screens reside, with the colors of each block representing the metric difference between the successive cocktails in that particular location on the plate. Each square unit of color corresponds to the comparison between the cocktails in the successive generations in that location on the plate. In the top, the C6 metric is used while the <i>CD_coeff</i> is shown below. Both metrics were able to highlight two rows of cocktails that were altered considerably between generations 8 and 8A, in the form of a line of darker wells in the lower third of figures. The C6 metric, however, identified that cocktails outside of these two rows were slightly different, when they were actually identical. This discrepancy most likely arises from the C6 metric's use of penalties in its PEG and salt terms.</p

Heat map for (A) the C6 metric (B) the <i>CD_coeff</i> and (C) the <i>CD_coeff</i> focusing only on the cocktail fingerprint term for the PEG molecular weight screen.

<p>All cocktails in this screen are identical, except for the PEG component, which samples ten of the molecular weight PEGs used in the standard 1,536 crystallization screen in our laboratory. The cocktails are ordered by increasing PEG molecular weight which makes the trend clear.</p