Optimization to the SAXS data and suits here as a tool to cross-validate the results obtained with molecular modeling. The three different approaches to SAXS data analysis will in concert address the different degrees of freedom in the complex protein samples. According to the basic SAXS analysis, MALS and AUC analyses, a distribution of primarily smaller oligomers exists when screening the solution behavior of GACwt protein at different concentrations, however complemented by a small fraction of larger oligomers. Earlier reports based on TEM images and our own TEM data indicate that higher order oligomers form as elongated species. Hence, a tentative octamer and a hexadecamer model were constructed in accordance with what was learnt from the TEM measurements. Since the program EOM allows to model oligomers, we included dimers, tetramers and octamers in the starting pool. Good fits to the experimental data were obtained with chi MK-1775 Wee1 inhibitor values ranging from 1.2 to 1.7. The selected ensembles containing dimers, tetramers and octamers suggest that compact conformations are never or rarely selected. It can hence be concluded that the samples contain significantly flexible and rather extended species of dimers, tetramers and octamers. Figure 2a depicts the relative distribution of dimers, tetramers and octamers as given by the EOM analysis. Representative structures were carefully chosen among those most frequently selected by EOM and used as a starting pool for the complementary analysis with the program OLIGOMER for the GACwt data. The results obtained hence crosscheck the oligomer distribution given by EOM and SASREFMX. Within the given concentration range the analysis of GACwtCS data reveals a development in the distribution of oligomers where the level of tetramers increases with concentration up to 85% before the formation of octamers finally slightly depletes the tetramer pool. Accordingly, the presence of PR-171 dimers decreases as the concentrations are increasing. Fits between the experimental data and selected EOM generated structures are shown in Figure S12 in File S1 and furthermore a plot is shown in Figure S13 in File S1 showing the fits obtained from EOM, OLIGOMER and SASREFMX analysis of the GACwtCs data. The chi values range from 1.3 to 2.3. In the analysis no monomers are selected, consistent with the EOM result. The overall parameters extrapolated using SAXS, MALS and AUC data from the GACwt phophate-screen revealed the presence of significant amounts of higher order oligomers and EOM analysis can therefore not be carried out. This confirms and elaborates on the result from the EOM analysis and from the basic SAXS analysis. The appearence of octamers starts at a lower concentration compared to what was seen for GACwtCS. Again, the chi values are high, and based on the MALS analysis, it is suggested that the discrepancies in the fits are due to the presence of even larger species in solution. During the phosphate screen the basic SAXS data analysis shows a rapid shift to very large oligomers. For the OLIGOMER analysis the earlier mentioned selected structures were included. As for the GACwtPS a shift in the distribution is seen when increasing from 25 mM to 50 mM inorganic phosphate in the solution. Below 50 mM Pi primarily dimers and tetramers are found in the solution. Above that, octamers and 16-mers are present. This shift correlates very well with the shift seen in the parameters obtained from the basic SAXS analysis. When comparing the oligomer distribution derived from EOM analysis of the four datasets the results of the two pi screens seams more coherent than the results of the two concentration screens. As larger oligomers are induced by the presence of phosphate and hence larger changes in the oligomer distribution are seen over the analyzed Pi concentration ranges.