Fig. 1: Structural and biochemical analyses of the PDCD4–ribosome complex.

a Schematic showing the ___domain architecture of the human PDCD4 protein. b Human HEK293T cells were either subjected to 24 h of glucose starvation or left untreated. Subsequently, the cytoplasmic lysates were subjected to fractionation on a sucrose gradient ranging from 10% to 40%. PDCD4 antibody was used to detect the distribution of the endogenous PDCD4 over different ribosome populations. Intensity, normalized to a maximum of 1.00, was calculated using ImageJ software. c Cryo-EM maps of PDCD4–40S (left), PDCD4–eIF3G–40S (middle) and PDCD4–43S (right). The composite maps shown are derived from multi-body refinement and after local resolution filtering using either Relion (PDCD4–43S) or DeepEMhancer (PDCD4–40S and PDCD4–eIF3G–40S). The density of the PDCD4–eIF4A complex in the PDCD4–eIF3G–40S map is shown at the lower contour level. The label of the PDCD4–eIF4A complex is boxed out, indicating that it represents only a putative model. d Molecular model of the PDCD4–43S complex highlighting the positions of the PDCD4–eIF4A complex and eIF3G (blue). Helix 18 and helix 34 of the 18S rRNA are colored in yellow, while uS3 and uS5 are represented in blue and green, respectively. e Cryo-EM map of the PDCD4–43S state filtered according to its local resolution. A zoomed insert highlights the closed “latch” region (red circle) within the mRNA channel, in contrast to the open latch in the 43S PIC “State III”. f Overview of the interactions of PDCD4 RBR with the mRNA channel. The 40S is shown as a colored density map derived from the PDCD4–43S state, while the RBR model is shown as sticks fitted into density (transparent) and colored in rainbow. The complete sequence of the RBR is also shown on the right. g–i Detailed interactions between RBR and the 40S subunit: R102 stacks with base C1701, and R103 interacts with the 18S rRNA backbone (g); R110 stacks with base C1698 (h); W124 inserts into a hydrophobic pocket in uS3 (i). j PDCD4 spatially clashes with the mRNA in the 48S IC (PDB: 7QP7). The mRNA entry channel is indicated by red dashed lines. k A region (amino acids 148–159) of PDCD4 forms an antiparallel β-sheet with the RRM ___domain of eIF3G (blue). l The overall conformation of the PDCD4–eIF4A complex in the PDCD4–43S state. m Zoomed view highlighting the direct contact between MA3c of PDCD4 and eIF3I (purple). The fitted model for eIF3I is shown to better illustrate its position. n, o Co-IP experiments were performed in human HEK293T cells transiently transfected with PDCD4-Flag and its mutants using anti-Flag beads. Interactions between PDCD4-Flag and the 40S ribosome (n) or eIF4A (o) were detected by immunoblotting for uS5 and eIF4A, respectively. CTRL: Control. The PDCD4 protein was detected by immunoblotting for the Flag tag. p Proposed model for PDCD4-mediated inhibition of 43S PIC assembly.