Azubel et al.Supporting material1

Supplemental Figure S1.The Native Spliceosome Is a Functional Splicing Complex

The figure shows that glycerol gradient-purified native spliceosomes restored splicing activity to a HeLa cell splicing extract (CILBIOTECH, Mons, Belgium) that had been inactivated by treatment with micrococcal nuclease. The splicing substrate used was -globin pre-mRNA, and three time points (0, 60, and 120 min) are shown for each experiment. Lanes 1–3, splicing activity of a HeLa nuclear extract (NE) in a splicing in vitro assay. After treatment with micrococcal nuclease the extract (NE+MN) was no longer active in splicing (lanes 4–6). Addition of gradient-purified native spliceosomes (nS) restored splicing activity to the inactivated extract, as can be judged by the appearance of the intron lariat intermediate (lane 9). Schemes of the pre-mRNA, and the splicing products and intermediates are shown on the right.

Robustness of the Common-Line Procedure

The common-lines procedure (Penczek et al., 1996) was run fourteen times. Each time, different approaches for alignment and classification were performed, as well as different selection of class averages (or the number of them). In order to compare between the resulting structures, the correlation coefficients between them were calculated, and are shown in the supplementary material (S-Table 1). In some cases, the solution had to be mirrored for the best correlation coefficient. As it could be seen from S-Table 1, in all cases, the values of the correlation coefficients obtained were at least 0.79, and in most cases higher than that. In addition, S-Figure 2 shows that the Euler angle assignments for the class averages used in the final 3D reconstruction cover the full angular range.

Further evidence for the robustness of the 3D structure is the reconstruction calculated from ~3000 negatively-stained particles, resulting in a structure having the same dimensions and low-resolution features as the model presented here (data not published).

Supplemental Figure S2. Euler Angles Distribution

A plot of Euler angles assigned to the 188 class averages used for the 3D reconstruction. Each class average is represented by a point in a coordinate system.

Supplemental Table S1. Correlation Coefficients between Reconstructions Obtained by the Common Lines Method

2* / 0.88
3 / 0.83 / 0.82
4** / 0.89 / 0.86 / 0.91
5 / 0.91 / 0.86 / 0.90 / 0.90
6 / 0.91 / 0.86 / 0.89 / 0.98 / 0.98
7 / 0.81 / 0.81 / 0.99 / 0.90 / 0.87 / 0.87
8 / 0.83 / 0.82 / 0.98 / 0.91 / 0.90 / 0.89 / 0.99
9* / 0.89 / 0.85 / 0.88 / 0.95 / 0.95 / 0.95 / 0.87 / 0.88
10* / 0.90 / 0.86 / 0.88 / 0.96 / 0.97 / 0.97 / 0.87 / 0.88 / 0.94
11 / 0.90 / 0.86 / 0.89 / 0.98 / 0.98 / 0.98 / 0.88 / 0.89 / 0.96 / 0.97
12 / 0.90 / 0.86 / 0.89 / 0.98 / 0.98 / 0.98 / 0.88 / 0.89 / 0.95 / 0.97 / 0.90
13 / 0.91 / 0.86 / 0.89 / 0.97 / 0.98 / 0.98 / 0.87 / 0.89 / 0.95 / 0.97 / 0.98 / 0.98
14 / 0.83 / 0.79 / 0.79 / 0.88 / 0.90 / 0.91 / 0.79 / 0.79 / 0.88 / 0.90 / 0.90 / 0.90 / 0.90
1 / 2* / 3 / 4** / 5 / 6 / 7 / 8 / 9* / 10* / 11 / 12 / 13

*Some solutions had to be mirrored.

**Initial model for the presented final reconstruction.

Supplemental References

Penczek, P.A., Zhu, J., and Frank, J. (1996). A common-lines based method for determining orientations for N > 3 particle projections simultaneously. Ultramicroscopy 63, 205-218.