Figure 5: SAXS reconstitution assay of the gp5/trx/gp4D complex.
(a) Schematic view of the experimental design. Samples containing proteins A and B are premixed in various ratios and placed in the sample cell. Rg values were extracted from the SAXS data. Increased Rg values indicate that a higher-order species is formed. Intermediate Rg value from the individual proteins in the mixture is indicative of non-interacting species. (b,c) Small-angle X-ray scattering (SAXS) curves of gp4D (5 μM) premixed with increasing amounts of gp5/trx (0, 2, 4, 8, 12 and 24 μM). Raw SAXS data (b) and the corresponding Guinier plots (c) for every sample. Colours indicate a gradual increase in the gp5/trx concentration (red to yellow). (c) The Rg values for the complex formed derived from these data and determined using Guinier plots. The insets in both b,c represent the theoretical SAXS curves of linearly combined spectra (from the available crystal structures) of the putative complex and the free proteins in solution. The data represent a complex between hexameric gp4D and gp5/trx in a molar ratio of 1:2, respectively. The theoretical SAXS curves of gp4D bound to gp5trx in a molar ratio of 1:1, 1:2, 1:3 and 1:4, respectively, are presented in Supplementary Fig. S4. (d) Scattering intensities (I0) shown as a function of concentration of gp5/trx. Data present scattering at zero angle of gp4D (5 μM) and increasing amounts of gp5/trx (0–24 μM), corresponding to the amount of electron scatterers at any sample. (e) SAXS results plotting the radius of gyration (Rg) against gp5/trx and gp4D ratios. Experimental SAXS data were collected for gp4D (5 μM) and increasing amounts of gp5/trx (0–24 μM) in a buffer containing 20 mM Tris-HCl (pH 7.5), 50 mM potassium glutamate and 2 mM DTT. Gp4D was premixed with 6.6 μM 15-mer ssDNA and 0.5 mM β, γ methylene dTTP to form hexameric molecules. Rg serves as an indicator for the formation of higher-order protein complexes. The dashed line represents a theoretical curve of Rg values that would be obtained if the components did not interact.
