s2 fig  (Thermo Fisher)

Journal: Nature Communications

Article Title: The cotranslational cycle of the ribosome-bound Hsp70 homolog Ssb

doi: 10.1038/s41467-025-67685-6

Figure Lengend Snippet: a Domain structure of Ssb. Nucleotide binding domain (NBD) with ATPase activity, and substrate binding domain (SBD), which is subdivided into a β-sheet domain (SBDβ) and an α-helical lid domain (SBDα). Ssb-NBD (light teal), Ssb-SBDβ (forest) Ssb-SBDα (split pea), linker region (gray). b Cryo-EM maps and atomic models of Ssb-ADP S1 and Ssb-ADP S2. RNCs arrested on truncated mRNA in complex with peptidyl tRNA Met-e and ADP-bound Ssb in two conformations termed S1 and S2. c Cross section through the cryo-EM map of Ssb-ADP S2. Densities of the nascent chain, mRNA and tRNA Met-e are highlighted. Insets show overlays of cryo-EM densities and atomic models of the nascent chain inside the exit tunnel and tRNA-mRNA base pairing at the peptidyl transferase center. d Binding of the nascent chain to the peptide-binding cleft of Ssb-SBDβ. Cross section through lowpass filtered (sdev=2.5), non-post-processed maps of Ssb-ADP S1 and S2. e Close-up of the contacts of Ssb-ADP S1 with the tunnel exit region. A detailed evaluation of contacts is provided in Supplementary Note and Supplementary Fig. . f Positioning of the Ssb-SBD relative to the ribosomal tunnel exit region. Top view of Ssb-ADP S1 (Ssb-SBDβ forest, Ssb-SBDα split pea) and Ssb-ADP S2 (sand). The NBDs, not resolved in the cryo-EM maps, were generated by superimposition of Ssb-ADP S1 (transparent light teal) and S2 (transparent sand) with the structure of Ssb-ADP predicted by I-TASSER based on PDB 2KHO (see Methods) for illustrative purposes. The inset shows sides views of Ssb-ADP S1 and S2. Color code of ribosomal proteins and rRNA: Rpl4 (tv orange), Rpl17 (olive), Rpl25 (light magenta), Rpl35 (violetpurple), Rpl39 (deepblue), Rpl26 (marine), Rpl19 (slate blue), Rpl31 (sky blue), helix 59 expansion segment 24 (ES24, pink), 5.8S rRNA (5.8 S, salmon).

Article Snippet: The resulting maps were post processed via DeepEMhancer using the ‘high-res’ weights provided by the developers resulting in cryo-EM maps Ssb-ADP S1 and S2 (Fig. ).

Techniques: Binding Assay, Activity Assay, Cryo-EM Sample Prep, Generated

Journal: Nature Communications

Article Title: The cotranslational cycle of the ribosome-bound Hsp70 homolog Ssb

doi: 10.1038/s41467-025-67685-6

Figure Lengend Snippet: The ribosomal protein Rpl25 (stage 1), serves as the primary binding site for Ssb-ATP (stage 2, Fig. ). h24 of the 25S rRNA serves as a primary binding site for RAC , (stage 3). At stage 3, Ssb-ATP and RAC bind without steric clashes; the Ssz1-SBDβ is near the tunnel exit , and the NBDs of Ssb and Ssz1 are prepositioned for complex formation (Supplementary Movie ). The emerging nascent chain first binds to Ssz1-SBDβ, displacing the Zuo1-LP motif from Ssz1-SBDβ . Freed from its contact with Ssz1, the Zuo1-ND exhibits significant flexibility . This allows coordinated conformational changes leading to the formation of the Ssb-ATP⋅RAC complex, in which Ssb-ATP interacts with Ssz1 . The Zuo1-ND extends and the Ssb-ATP⋅RAC complex rotates along the ribosomal surface, leading to positioning of Ssb-SBDβ at tunnel exit and handover of the nascent chain from Ssz1-SBDβ to Ssb-SBDβ (Supplementary Movie ). In the pre-hydrolysis state, the Zuo1-ND is fully extended, and the Zuo1-JD is positioned to trigger ATP hydrolysis by Ssb (stage 4 and Supplementary Movie ). ATP hydrolysis triggers conformational changes within Ssb , that disrupt its interaction with the Ssz1-NBD and enable RAC to return to its prior conformation (stage 5 and Supplementary Movie ). In stage 5, Ssb-ADP S1 is bound to the nascent chain (Fig. ) and has regained its contact with Rpl25 (Fig. ). The contact with Rpl25 is maintained as Ssb-ADP adopts the S2 conformation (Supplementary Fig. ). Ssb-SBDβ is now closer to the ribosomal surface and clashes with Ssz1-NBD, which leads to RAC release (stage 6, Supplementary Movie ). Ssb-ATP (stage 2 and 3): Supplementary Fig. ; RAC (stage 3 and 5): PDB 7X3K ; Ssb-ADP S1 (stage 5): Fig. , Ssb-ADP S2 (stage 6): Fig. , Ssb-ATP⋅RAC (stage 4): AF3-predicted model (Supplementary Note , Supplementary Fig. , and Methods). Large ribosomal subunit (PDB 6T7I, light gray), Zuo1 (orange), Zuo1-JD (limon), Ssz1-NBD (yellow orange), Ssz1-SBDβ (sand), Ssb-NBD (light teal), Ssb-SBDβ (forest) Ssb-SBDα (split pea), Rpl25 (light magenta).

Article Snippet: The resulting maps were post processed via DeepEMhancer using the ‘high-res’ weights provided by the developers resulting in cryo-EM maps Ssb-ADP S1 and S2 (Fig. ).

Techniques: Binding Assay