Reaction of Tgl with C30 and <i>ab initio</i> model of C30.

A: Tgl, at the indicated concentrations, was incubated with purified C30 (at 25 μM; see also S6A Fig) and samples of the reaction mixture collected at the indicated times. The samples were mixed with loading dye and boiled to stop the reaction and resolved by SDS-PAGE. The level of C30 decreases over time as the result of Tgl-mediated cross-linking into higher molecular weight forms (see S5A Fig for representative SDS-PAGE gels). Tgl activity is expressed as the level of C30 in each time point, normalized to the initial amount of the protein. B: The assays were conducted as described in A, except that Tgl (at 8 μM) was incubated with different concentrations of C30, as indicated (see S5B Fig for representative SDS-PAGE gels). C: Tgl (at 4 μM) was incubated alone during 20 min and C30 added at this point, to a final concentration of 25 μM. Samples were taken along time and analyzed by SDS-PAGE (representative gels are shown in S5C Fig). The level of Tgl in each sample was quantified and normalized to the initial amount of protein. In A-C, experiments were repeated three times with different preparations of Tgl and C30. D: SAXS intensity of C30 (blue circles), I(s), is represented in logarithmic scale as a function of the momentum of transfer, s. The inset shows the SAXS-generated ab initio molecular envelope obtained by clustering and averaging models from 20 independent runs, with a Normalized Spatial Discrepancy (NSD) of 0.8 Å. The low-resolution reconstruction of C30 in solution is compatible with a high-order macromolecular assembly, most likely a hexamer, termed (C30)6. Both mesh and solid representations of the model are shown, both centered at the origin of the cartesian axis. The red line corresponds to the scattering profile calculated from the ab initio model that best fitted the experimental data (χ2 = 2.2). Point-by-point residuals of the fitting and the absolute intensities are shown in the bottom panel. E: Pair-wise distance distribution (P(r)) of SEC-purified C30 calculated from data in the range 0.010s-1 (see the Material and Methods for details). The derived Rg and Dmax values are displayed in dashed lines.