Focused Classification of mRNA Exit Channel

A similar approach as for the mRNA entry channel (see section 3.3.4.4) was performed for the area around the mRNA exit channel. Here, low-resolved and quite heterogeneous density clearly indicated the presence of additional factors or at least factor parts. A smeared out structure was visible connecting the eIF3 core domain around the solvent side of the 40S all the way to eIF3b. Thus, a local mask was created including only this flexible area (figure 3.22-E; also see figure S4 for a detailed representation of the eIF3 core structure). All other parts of the complex — the vast majority of the density — were subtracted from the particle projection images to minimize their influence during 3D classification. Local classification was performed resulting in ten classes of which three showed well resolved density (see figure 3.22-B/C/D).

Especially the first class that was found (figure 3.22-B) showed significantly more density than the consensus refinement. This additional density was not occupied by any of the previously fitted proteins or ribosomal components and therefore must be attributed to something else. Its shape did not show obvious resemblance with any of the thus far unfitted proteins. Furthermore, the search for cross-links in this area, formed by either the ribosomal proteins or any of the other eIF factors already present, did not give any hints about the origin of the additional density. Nevertheless, three parts were showing cross-links: a small density above eIF3b, eIF3b itself, and the already modeled components RACK1 and eIF3d above the mRNA exit channel.

The small density close to eIF3b was attributed to the RNA binding domain of eIFg, based on cross-links found for eIF3g with the adjacent the ribosomal proteins (figure 3.19; cyan

Fig. 3.22: Local classification of the mRNA exit channel. Very little density is visible when investigating the area near the complex’s mRNA exit channel (panel A). However, when increasing the threshold, a large amount of spiky, dynamic density shows up from the void (panelE). To get a better understanding for this area enclosed by the green mask (panelsE andF), signal subtraction and 3D classification were performed. As a result, three interesting classes were identified (panelsB–D), which all show additional, but still quite fuzzy density.

Furthermore, a density above the WD40β-propeller of eIF3b (blue) was found and attributed to eIF3g (green) according to cross-linking mass spectrometry data.

3.3eIF3inContextofthe48STranslationInitiationComplex81

Fig. 3.23: Cross-links found for proteins at mRNA exit channel. At the 48S-IC mRNA exit channel, a heterogeneous, but connected density was identified (figure 3.22-E). Although not stable enough for straightforward model building, the cross-links found for this part of the 48S-IC were helpful to locate the position of several factors. All cross-link forming lysines are shown in red. The Apanels show eIF3d (pale yellow) and RACK1 (cyan) and their respective cross-links with each other and the 40S ribosomal proteins 28 and 16 (figure 3.19; blue lines). RACK1 shows cross-links on one side with with eIF3d (A.3) and on the opposite site with eIF4B, for which no density was visible. For eIF3b, the connection to 40S ribosomal protein 9 is shown (panelB.1), as well as a possible connection to eIF3i (panelB.2), although no density is visible that could have been attributed to it (figure 3.19;

green lines). TheC panels show the interaction between the RNA binding domain of eIF3g and 40S ribosomal protein 3 (figure 3.19; cyan lines).

connection). Additionally, a part-structure of eIF3g from the PDB was fitted into the density (figure 3.23-C.1).

Although the general position of the nine-bladed eIF3b WD40 β-propeller was already identified using the refined structure, the cross-linking experiment gave more detailed insights (figure 3.19; green connections). Here, cross-links with both ribosomal proteins and eIF3i were found (see figure 3.23-B), helping to identify the exact orientation of the eIF3b and giving an indication about where eIF3i is most likely to be found (see figure 3.23). Judging from the orientation of the cross-links, eIF3i could easily fit into the large circular density of the first class from 3D refinement (figure 3.22-B), left of eIF3b in the picture.

Directly above the mRNA exit channel, the two proteins RACK1 and eIF3d were already fitted into the refined density of the 48S-IC (figure 3.23-A.2). However, an assessment of the cross-links found for both these proteins (figure 3.19; blue connections) not only proved the fitted orientations to be correct, but also revealed evidence regarding plausible locations of eIF4B and eIF4A: lysines on the solvent side of RACK1, pointing towards the unstructured area that was previously enclosed by a mask for 3D classification, ap-parently form cross-links with eIF4B. Therefore, eIF4B was located close to the mRNA exit channel, being a part of the flexible density within the 3D mask, although not di-rectly visible after refinement. Unfortunately, the local classification did not reveal any additional density that could have been attributed to eIF4A or eIF4B.

Apart from the factors already mentioned, probably a lot of the flexible density surround-ing the mRNA exit channel is composed of parts from the eIF3 core subunits. This is suggested by the cross-link mass spectrometry results, since the eIF3 core, particularly eIF3a and eIF3c, shows connections to most of the other complexes bound to the solvent side of the 40S (see supplementary table S2 and figure S4). eIF3a forms cross-links with eIF3b, c, d, f, g, h, m, and also eIF4B. For eIF3c, there are cross-links with eIF1, eIF2γ, eIF3a, d, e, h, and l listed.