Cryo-EM density maps of the 40S subunit and MFC. (A) The 40S subunit reconstruction (6,267 particles; resolution 19 Å) contoured at 1.5σ in three orientations, with the atomic coordinates of the yeast 40S subunit (14) fitted: head rRNA, blue; platform, green; body, red; and helix 44, cyan. The small subunit proteins are shown in magenta. Three orthogonal views about axes as marked are shown, and major structural landmarks are labeled. (B) Close-up, sectioned views of the head–body/platform connections at a contour level of 1.5σ in the same orientations and with the same color scheme as in A (thumbnail images indicate the directions of view). (C) Views of the 40S subunit reconstruction after computational segmentation, with the density for head colored blue and the platform and body together colored yellow. (Scale bar, 50 Å.) (D) Orthogonal views of the MFC reconstruction (19,446 particles, 14-Å resolution). The scale is identical to that of A and C. (Scale bar, 50 Å.)

Analysis of the 43S complex. (A) Orthogonal views of the platform/body region of the 20-Å 43S reconstruction (as in Fig. 3A) with the platform and body colored as in 2C and the MFC colored magenta, oriented such that the body is in the same orientation in each image as in Fig. 2C. Thumbnails show the 40S platform and body aligned by reference to the body of the 43S for each view. Thumbnails are 50% of the main image size. (B) Equivalent views to A in which the 43S density corresponding to the reordered small subunit is colored gray (using the head from 3B for the head and the 20-Å structure (A) for the body/platform) fitted with the atomic models for the head, platform, and body (13), colored blue, green, and red. The MFC density is shown as a mesh surface, colored magenta. Thumbnails of the MFC within the 43S (semitransparent surface) superimposed on the isolated MFC reconstruction (magenta mesh) are shown in each orientation. Asterisk, helix 18. Thumbnails are 75% of the main image size. (C) Schematic diagram of the conformational changes observed here in the 40S subunit (Left, inactive) on binding the eIFs that form the MFC (Center). The head is colored blue and the platform/body is yellow in each case, as in Figs. 2C and 3, with arrows indicating the domain movements after eIF binding that release the subunit head. (Center) The head displays movement along with the covalent link between it and the body. (Right) The small subunit has reverted to its usual structure within the elongating ribosome with the binding of the large 60S subunit. h, head; p, platform; b, body; mfc, multifactor complex.

Reconstructions of the 43S complex. (A) The main figure (in yellow/blue) depicts orthogonal views of the 43S reconstruction at 20-Å resolution, incorporating 27,101 of 31,756 images. The head (h), body (b), and platform (p) are labeled. The head is colored blue, and the rest of the reconstruction is in yellow. The left-hand image is of the reconstruction in gray mesh with the bootstrap variance map computed from its data set displayed at 1.7σ (green) and 1.77σ (red) (the maximum peak in each case was 2σ). (B–D) Equivalent orthogonal views to those shown in A for three subclass reconstructions of the 43S, displaying the variable position adopted by the head. B was calculated from 6,864 particles, C from 8,269 particles, and D from 9,467 particles. In each case, the resolution of the map is 30 Å. See SI Fig. 7 for representative class averages and 2D variance maps for these subclasses of 43S particles. See SI Fig. 8 for 3D variance analysis of these data; the variance map for the first orientation displayed is shown on the left of each figure, as in A. (E) Orthogonal views of the head regions of the reconstructions shown in B–D superposed (B, green; C, blue; and D, red). On the right (upper thumbnail) is the atomic structure of the head of the ribosome from the T. thermophilus crystal structure (21) (red) superimposed with reference to its body on that of the E. coli crystal structure (15) to demonstrate crystallographic evidence for the modes of head movement observed between the 40S and 43S structures. The lower three thumbnails show the 40S head aligned on its body to the 43S for each view. The thumbnails are 50% the size of the main images.

Preparation and hydrodynamic behavior of 40S complexes (see SI Materials and Methods for full details). (A) eIFs, 40S, and 40S-MFC (43S) complex loaded onto an SDS/PAGE gradient gel. A few of the eIF proteins show very similar electrophoretic behavior to 40S proteins and thus generate multiple bands (marked by asterisks) in the 43S lane. The 43S complex loaded here was isolated as fractions (see vertical dotted lines in B) from a sucrose-density gradient. (B) A 254-nm absorbance elution profile from a sucrose-density gradient; the presence of the eIFs in the 43S sucrose-gradient peak was routinely confirmed by means of Western blotting using the antibodies defined in SI Materials and Methods. (C) The 40S (gray, circles) and the 43S complex (red, diamonds) were subjected to sedimentation velocity analytical ultracentrifugation, yielding apparent sedimentation coefficient distributions. These were fitted with Gaussian equations (lines), giving sedimentation coefficients of 36.4S for the small subunit and 41.5S for the preinitiation complex. Corrected for finite solute and solvent effects on the basis that the small subunit value is 40S, the preinitiation complex value becomes 45.6S. (D) Assembly of complexes between 40S and MFC eIFs. PhosphorImages of native gels loaded with 40S–eIF complexes incorporating [³⁵S]Met-tRNA_i and GMP-PNP. The addition of eIF3 leads to a shift in the ribosome complex band (left-hand side). The further addition of eIF5B and the 60S subunit led to a further shift, associated with the formation of 80S (right-hand side). (E) A typical experiment following the kinetics of methionyl-puromycin synthesis by 80S complexes prepared by using the components shown in A plus 60S, eIF5B, methionyl-tRNA_i, and GTP (instead of GMP-PNP). A PhosphorImage of samples from a typical experiment that were allowed to run on cation exchange TLC is shown. (F) Time course of formation of the ³⁵S-labeled methionyl-puromycin product, taking the samples featured in E plotted against time. The y axis is calibrated in relative signal intensity. Also plotted are data from an experiment in which eIF5B was omitted.