For the SAXS measurements a microfluidic setup for data collection was applied enabling screening of the solution behavior of GAC in response to different experimental conditions. Also, the use of the microfluidic setup enabled the study of a timedependent oligomerization effect of the protein. The crystal structures of mouse GAC and human KGA with and without substrate have recently been published; however, it has never been possible to determine the structure of the N- and the C-termini. Here we have used SASREFMX to identify if the known tetrameric structure is compatible with the solutions investigated in this study. In SASREFMX, polydisperse solutions containing partially dissociated assemblies, can be analyzed by simultaneous fitting of the scattering data from a concentration range of the protein species in equilibrium whereby the outcome is a rigid body model of the whole macromolecule and its volume fraction at each concentration. From the Rg and MW values we estimated that the GACwtCS samples include mainly dimeric and tetrameric protein with the addition of higher oligomers in the most concentrated samples. Hence, the program was applied on this dataset both including and excluding the highest concentration measurements. The resulting tetrameric model including all data yielded chi values ranging from 1.1 to 2.9, whereas, by excluding the highest concentration, the highest chi value was reduced to 1.4. The typical models calculated in both scenarios were rather similar. The volume fractions of dimers and tetramers are shown in Figure 2a. The models calculated excluding the highest concentration data curves were preferred since the high concentration scattering curves could potentially include structural information that was not present in the lower concentration scattering curves. The low-resolution solution structure and fits to the experimental data for three of the seven included scattering curves are shown in Figure 3. Volume fractions and translations are listed in Table S1 in File S1 for both described models. See materials and methods for details. To verify the presence of different oligomeric species in equilibrium, as suggested by the basic SAXS analysis, we next subjected our proteins to MALS and AUC studies. Indeed the presences of several species were verified by both methods. Due to differences in experimental conditions when applying the different methods, results are not exactly quantitatively AZ 960 comparable. Within the concentration range, measurable by MALS, three different species could be detected, but no significant changes in the distribution of oligomers were noticeable as a function of the protein concentration. The SASREFMX rigid body high throughput screening modeling analysis confirmed that the previously known tetrameric structure with addition of the Nand the C-termini is supported by our SAXS data. Therefore, we proceeded to a more thorough approach for describing the flexible C- and N-termini using the program EOM. In accordance with experimental data, the program selects ensembles of theoretical scattering curves generated from very large 
pools of structures, where the assumed flexible parts of the protein are in random conformations. The selected pool of structures does not describe the actual combination of specific structures that is found in the solution, but rather a collection of structures representative of the solution state in size and conformation. As a complementary approach to the SASRFMX and EOM analysis we also employed the program OLIGOMER to evaluate the quaternary structure distribution. From a predefined set of structures of different size and shape, the program calculates a linear combination of the corresponding scattering curves, in accordance with the experimental data. This hence yields the volume fractions of each oligomeric species.