Supplementary Figure 7: Preparation and cryo-EM of the native 40S–ABCE1 complex.
From: Structure of the 40S–ABCE1 post-splitting complex in ribosome recycling and translation initiation
a, The TEV-eluate of affinity purified tandem-affinity tagged ribosome–ABCE1 complexes was applied to a 5-30% sucrose density gradient and the A260 profile was recorded. b, Fractions containing 40S subunits and ABCE1 were pooled and analyzed by SDS-PAGE. Proteins were identified by mass spectrometry. Notably, in addition to ABCE1 and 40S proteins (RPS), the complex also contains components of the 43S pre-initiation complex (eIF2α and eIF2γ, eIF3c and eIF3j) together with the fatty acid synthase as a common contaminant of 40S preparations. c, Samples were subjected to cryo-EM and single-particle analysis. The fatty acid synthase could be easily sorted out during 2D classification. d, After refinement, 3D classification was performed in RELION-2. In the first round three classes (63%) show a clear density for ABCE1 and two of them showed additional extra density emerging from the P-site. These classes (43.5%) were joined for a second round of classification. Here, four of five classes only differ in the appearance of the density in the P-site, which most likely represents initiator tRNA (tRNAi) in various positions. One class, however showed additional density in the position where eIF1A is located. This class (17.6%; 9,500 particles) was refined to a final resolution of 14 Å (e) according to the “gold standard” criterion (FSC = 0.143). The model of ABCE1 in the post-splitting state was fitted as rigid body without further adjustments into the ABCE1 density. eIF1A (blue) as well as tRNAi (green) could be identified by rigid body fitting of 43 and 48S-initiation complex structures (eIF1A from 4UER, ref. 55, tRNAi taken from 3JAP, ref. 40). f, A difference map was calculated between the native (left) and the in vitro reconstituted 40S–ABCE1 maps (middle). The difference map was superimposed to the in vitro reconstituted 40S–ABCE1 map (contoured at 3.5 σ). Notably, significant difference between the two maps (left and middle) occurred in the region of initiator tRNA (tRNAi) and eIF1A. No conformational differences were observed for ABCE1.
40 Llacer, J.L. et al. Conformational differences between open and closed states of the eukaryotic translation initiation complex. Mol. Cell 59, 399-412 (2015).
55 Aylett, C.H. et al. Structure of a yeast 40S–eIF1–eIF1A–eIF3–eIF3j initiation complex. Nat. Struct. Mol. Biol. 22, 269-271 (2015).
