The cotranslational cycle of the ribosome-bound Hsp70 homolog Ssb

Domain: chemistry
Published: 2026-01-16
Editorial outcome: minor_revision

Abstract
Coupling of ribosomal translation with cotranslational protein folding is essential for cellular homeostasis. In eukaryotes, Hsp70 and its J-domain cochaperone, the heterodimeric ribosome-associated complex (RAC), are central to this process; however, mechanistic insights into the coordination of Hsp70 function with translation remain limited. Here, we present two cryo-EM structures of the ribosome-bound yeast Hsp70 Ssb, identifying Rpl25/uL23 as the ribosomal binding site and revealing its interaction with a model nascent chain. Together with detailed biochemical and mutational analyses, these structures enable us to delineate the intricate RAC-dependent cycle, which positions the substrate binding domain of Ssb-ATP close to the tunnel exit to receive nascent chains. This arrangement allows Ssb to undergo substantial conformational changes upon ATP hydrolysis without steric clashes with the ribosome, while the substrate binding domain of Ssb, now anchored by the tightly bound nascent chain, remains close to the tunnel exit.

Introduction
Cotranslational processes - such as folding, complex assembly, and membrane translocation - critically rely on Hsp70 chaperones. Canonical Hsp70s cooperate with J‑domain (JD) cochaperones, which stimulate their ATPase activity and substrate binding, and with nucleotide exchange factors (NEFs), which facilitate ADP-release and rebinding of ATP1–4 (Supplementary Fig. 1a). Yeast Ssb is a canonical Hsp70 (encoded by the nearly identical SSB1 and SSB2 genes), consisting of an N-terminal nucleotide binding domain (NBD) and a C-terminal substrate binding domain (SBD) (Fig. 1a). The NBD possesses ATPase activity, the SBD is subdivided into a β-sheet domain (SBDβ) and an α-helical lid domain (SBDα) (Fig. 1a). As in other canonical Hsp70s, the two domains of Ssb are connected via a linker that allows allosteric coupling of ATP hydrolysis with tight substrate binding to the SBD (Supplementary Fig. 1a). When Ssb is bound to ATP, the linker interacts with the NBD, and the SBDα lid adopts an open conformation5. Upon ATP hydrolysis, significant structural rearrangements occur, which lead to the detachment of the linker from the NBD, allowing the SBDα lid to close over the substrate binding pocket on SBDβ, thereby stabilizing substrate binding1,4,6.

Ssb is critically important for cotranslational protein folding, as it binds directly to ribosomes via SBDα5,7 and, from this position, accesses newly synthesized polypeptides8–10. To perform its function, Ssb requires a unique cochaperone, termed ribosome-associated complex (RAC)6,11,12. The heterodimeric RAC consists of the JD protein Zuo113 and the Hsp70 homolog Ssz16,11. The Zuo1 subunit interacts with the 60S as well as 40S ribosomal subunit6,14–17 and possesses a unique domain structure6,16,17 (Supplementary Fig. 1b). Ssz1 is a non-canonical Hsp70 as it binds, but does not hydrolyze ATP, and lacks the C-terminal portion of SBDβ and the complete SBDα6,18,19 (Supplementary Fig. 1b). In the absence of ribosomes, Ssz1 and Zuo1 engage in a tight interaction, mediated by the N-terminal domain of Zuo1 and the rudimentary Ssz1-SBDβ6,18–20. However, when bound to ribosomes, RAC adopts a highly flexible structural arrangement, as evidenced by the markedly different conformations observed in ribosome-bound RAC through cryo-EM studies16,17. Conformational flexibility within RAC is highlighted by the extreme N-terminus of Zuo1, termed the LP-motif (Zuo1-LP; Supplementary Fig. 1b21), which binds to Ssz1-SBDβ as a pseudo-substrate but is displaced upon nascent-chain binding21. Displacement of the Zuo1-LP occurs as soon as the nascent chain emerges from the ribosomal tunnel, thereby loosening the Zuo1-Ssz1 interaction and permitting conformational rearrangements of the N-terminal Zuo1 domain (Zuo1-ND; Supplementary Fig. 1b). Shortly thereafter, the nascent chain is handed over from Ssz1-SBDβ to Ssb-SBDβ, but how this transfer is coordinated remains unknown21.

RAC possesses a mammalian homolog, termed mRAC, which consists of MPP11/DNJC222,23 and HSPA14/Hsp70L122,24. RAC and mRAC are strictly required for cotranslational Hsp70 function11,12,22,24,25. However, why Ssb function depends on such a complex JD partner - whereas Hsp70s typically cooperate with structurally simpler monomeric or homodimeric JD proteins26,27 - remains unresolved. A major obstacle has been the lack of structural information on Hsp70s bound to translating ribosomes and nascent chains17,28. In this work, we provide structural insights into how Ssb interacts with ribosomes and nascent chains, and elucidate the interplay between Ssb and RAC during cotranslational protein folding.

Results
Cryo-EM structure of ADP-bound Ssb associated with translating ribosomes
To determine the structure of Ssb associated with translating ribosomes, ribosome-nascent chain complexes (RNCs) carrying FLAG-tagged 3-phosphoglycerate kinase (FLAG‑Pgk1‑70; Supplementary Fig. 1c–f) were generated in a yeast in vitro translation system in the presence of Ssb, were subsequently purified by FLAG affinity chromatography, and were analyzed by cryo‑EM (Supplementary Fig. 2, see Methods). In these samples, ribosomes representing diverse states along the translational cycle were detected. Notably, non-rotated RNCs containing a P-site tRNA and associated with the density of a trailing ribosome displayed enhanced density definition near the ribosomal tunnel exit (Supplementary Fig. S3). Further analysis of these particles (Supplementary Fig. S2) yielded two cryo-EM structures in which Ssb was bound to ribosomes stalled in a non-rotated, post-translocation conformation with a P-site Met-elongator tRNA (tRNAMet-e). In both structures, the Ssb-SBD (Fig. 1b and Supplementary Table 1) was in the closed conformation characteristic for the ADP-bound state of Hsp70s, in which SBDα is tightly packed against SBDβ (see Introduction). The Ssb-NBD was not visible in the cryo-EM maps, consistent with the known flexibility between the NBD and SBD in ADP-bound Hsp70s4,29,30. The resolution of the complexes, termed Ssb-ADP S1 (2.8Å) and Ssb-ADP S2 (3.0Å), allowed precise visualization of tRNA-mRNA interactions, including Watson-Crick base pairing between tRNAMet-e and its cognate codon (Fig. 1b,c, Supplementary Fig. 1e, Supplementary Fig. 4a–h). The density of nascent FLAG-Pgk1-70 was well defined within the proximal region of the exit tunnel, allowing for side chain assignment of a large fraction of the 17 C-terminal residues and enabling confident modeling of the nascent chain (Fig. 1c and Supplementary Fig. 1e). While the nascent chain remained traceable through the constriction site formed by loops of Rpl17 (uL22) and Rpl4 (uL4) in the exit tunnel, its structural definition decreased progressively (Fig. 1c,d, and Supplementary Fig. 1f). Local resolution of the Ssb-SBD enabled side chain assignment for SBDα. In case of SBDβ, flexibility relative to SBDα and the translating ribosome hindered side chain assignment; therefore, the SBDβ backbone was modeled as a rigid body (Supplementary Fig. 4d,h). Ssb-ADP was attached to the ribosome mainly via SBDα with the peptide-binding cleft of SBDβ positioned close to the tunnel exit (Fig. 1e and Supplementary Fig. 5a, b). The inherent flexibility of nascent FLAG-Pgk1-70 bound to Ssb-SBDβ prevented direct visualization at high resolution, however, low-pass filtered non-sharpened maps revealed an extension leading away from the ribosomal tunnel exit to the SBDβ substrate binding cleft, which we tentatively attributed to the nascent chain (Fig. 1d and Supplementary Fig. 1f, red density).

The combined data suggested that both Ssb-ADP S1 and S2 corresponded to ribosome-bound Ssb engaged with a nascent chain substrate (Fig. 1b,d and Supplementary Fig. 1e,f). In both states, Ssb-SBD adopted the closed conformation of an ADP-bound Hsp70 (Supplementary Fig. 1a), but differed in the position of SBDβ relative to the translating ribosome, which was 19° closer to the ribosomal surface in S2 when compared to S1 (Fig. 1f and Supplementary Fig. 5a, b).

Previous studies5,7 employed crosslinking approaches and mutational analyses to identify contact sites between Ssb and the ribosome. These studies revealed that the most C-terminal helix of Ssb (Ssb-αD, residues 592-611, Supplementary Fig. 5c) is essential for ribosome-binding5,7. On the ribosomal side, Rpl35 (uL29), Rpl19 (eL19), Rpl39 (eL39)5, Rpl26 (uL24)28, and the 25S rRNA helix 59 expansion segment 24 (h59-ES24)5 were found to crosslink to Ssb. While in the atomic model of Ssb-ADP S1 the distance of the SBD from Rpl35, Rpl19, Rpl39, and Rpl26 was inconsistent with crosslinking, the position of the SBD in Ssb-ADP S2 reveals the precise molecular basis of these crosslinks (Fig. 1f, Supplementary Fig. 5a,b, and Supplementary Note 1). Only two ribosomal binding sites were shared by Ssb-ADP S1 and S2. One was the contact of Ssb-K567 and Ssb-R568 with h59-ES24 (Supplementary Fig. 5a,b). This contact provides the molecular basis for the observation that substituting K567-R568 with glutamates partly impairs Ssb´s ribosome-binding5 (Supplementary Fig. 5c). The other was the contact of Ssb-αD with Rpl25 (Fig. 1e and Supplementary Fig. 5a,b), which had not been identified previously.

Ssb interacts with ribosomal protein Rpl25 at the ribosomal tunnel exit
Analyses of the Ssb-ADP ribosome interface in the atomic models of states S1 and S2 revealed that Ssb primarily contacts Rpl25 through salt bridges and long-range ionic interactions (Fig. 2a and Supplementary Fig. 6a). Key contributions to the interface came from Ssb residues R596, K597, K603, and R604 (termed the Ssb-RKKR motif, Supplementary Fig. 5c) and Rpl25 residues E77, D131, D134 (termed the Rpl25-EDD motif) and the Rpl25 carboxyl terminus (Fig. 2a and Supplementary Fig. 6a). Specifically, salt bridges were observed between Ssb-R604 and Rpl25-D131, Ssb-R596 and Rpl25-D134, and between Ssb-K603 and the C-terminus of Rpl25 (I142). Long-range ionic interactions were formed between Ssb-K597 and Rpl25-D134, and Ssb-K608 and Rpl25-E77. Although the overall geometry of the binding interface did not differ substantially between S1 and S2, Ssb-SBDβ was closer to the ribosomal surface in S2 than in S1 (Fig. 1f and Supplementary Fig. 5a,b).

To validate the interface, we mutated the EDD-motif to three alanine (termed Rpl25-AAA) or three lysine residues (termed Rpl25-KKK) (Supplementary Fig. 6b) and compared ribosome-binding of Ssb to wild type and Rpl25-AAA/KKK ribosomes in total cell extracts. Consistent with earlier findings5, approximately 50% of wild type Ssb was ribosome-bound, with about two-thirds of this fraction exhibiting salt-resistant binding (Fig. 2b and Supplementary Fig. 6c). Ribosome-binding of Ssb was reduced more than 2-fold in extracts of the Rpl25-AAA and Rpl25-KKK strains under low- and high-salt conditions (Fig. 2b and Supplementary Fig. 6c). To further validate the role of the Rpl25 EDD-motif, we used recombinant Ssb and purified non-translating ribosomes (Supplementary Fig. 6d–f). Wild type Ssb was bound to non-translating ribosomes, however, in this case high-salt conditions led to complete ribosome-release (Fig. 2c,d). The observation suggested that salt-resistant ribosome-binding involved the interaction between Ssb and the nascent chain (see below). Binding of Ssb to purified Rpl25-KKK ribosomes was strongly reduced, while binding of RAC (control) remained unaffected (Fig. 2c). These data established Rpl25 as the primary ribosomal attachment site of Ssb and demonstrated that residues E77/D131/D134 in Rpl25 are crucial for the interaction. In retrospect, the absence of Ssb-Rpl25 crosslink products5,28 is evident: lysine residues are abundant in both Ssb and Rpl25, however, Rpl25 lacks solvent-exposed lysine residues in close proximity to Ssb-SBD.

The Rpl25-AAA and Rpl25-KKK strains exhibited severe growth defects (Supplementary Fig. 6b), exceeding the only minor growth defects of Ssb mutant strains with significant ribosome-binding defects5,7. A potential explanation emerged from Rpl25’s established role as a critical binding site for signal recognition particle (SRP), which requires this interaction for efficient cotranslational targeting31–34. Structural superposition of Ssb-ADP S2 with SRP-bound RNCs35 revealed that Ssb and SRP binding would be mutually exclusive (Supplementary Fig. 6g). We tested if targeting of the type II membrane protein Dap2, a well-characterized in vitro and in vivo substrate of SRP36,37, was affected by mutations in the Rpl25 EDD-motif. Indeed, expression of Dap2 in a Rpl25-KKK strain was significantly reduced when compared to wild type and resembled expression of Dap2 in a Δsrp54 strain (Supplementary Fig. 6h). Moreover, Ssb which was efficiently crosslinked to nascent Pgk1 (Supplementary Fig. 1d and Supplementary Fig. 6i), did not crosslink to nascent Dap2 under the same conditions (Supplementary Fig. 6i37). This pattern changed in the absence of SRP: under this condition Ssb was efficiently crosslinked to nascent Dap2 (Supplementary Fig. 6i). The data support a model in which SRP and Ssb compete for an overlapping ribosomal attachment site involving the Rpl25 EDD-motif. They further suggest that the competition of SRP and Ssb for nascent chains, observed in a global approach38, is unlikely to result from a general inability of Ssb to interact with SRP substrates, but may rather reflect direct competition at the tunnel exit. Importantly, the impaired delivery of the SRP-dependent ER model protein Dap2 (Supplementary Fig. 6h) provides a plausible explanation for the severe growth defects observed in strains carrying mutations in the Rpl25 EDD-motif (Supplementary Fig. 6b).

Rpl25 provides the binding site for Ssb in the ATP- and ADP-bound state
To further probe the electrostatic nature of the Ssb-Rpl25 interaction, we engineered a charge-reversal variant of Ssb (Ssb-DD1-DD2, Supplementary Fig. 7a) by replacing the basic residues of the RKKR-motif with aspartates (Fig. 3a). Analysis of Ssb-DD1-DD2 revealed a striking context-dependent binding pattern. In cell extracts containing actively translating ribosomes, Ssb-DD1-DD2 showed significantly reduced but detectable ribosome association (Fig. 3b,c). Under these conditions, Ssb-DD1-DD2 binding was fully salt-resistant, suggesting that the interaction was primarily mediated by the nascent chain (Fig. 3b,c). Consistently, with purified non-translating ribosomes, binding was completely abolished (Supplementary Fig. 7b and Fig. 2d), highlighting the contribution of nascent chains to ribosome-binding of Ssb.

The combined data revealed that replacing either the positively charged Ssb RKKR-motif with negatively charged residues (Fig. 3b,c) or substituting the negatively charged Rpl25 EDD-motif with positively charged residues (Fig. 2b,c) exerted a similar effect on ribosome-binding of Ssb. We thus performed a charge reversal binding experiment with recombinant Ssb-DD1-DD2 (Supplementary Fig. 6f) and non-translating Rpl25-KKK ribosomes (Supplementary Fig. 6e). Indeed, binding of Rpl25-KKK to Ssb-DD1-DD2 was improved when compared to binding of wild type Ssb confirming the electrostatic nature of the interaction (Fig. 3d). Thus, the Rpl25 EDD- and Ssb RKKR-motifs were not only proximal to each other, as shown in the atomic models Ssb-ADP S1 and S2 (Fig. 2a and Supplementary Fig. 6a), but also essential for Ssb´s direct association with ribosomes.

It has been proposed that Ssb, particularly in its ATP-bound state, does not bind directly to the ribosome but is instead recruited through the dimerization of its NBD with the NBD of Ssz12,28. However, in total cell extract, an ATPase-deficient Ssb mutant was bound to ribosomes, albeit less efficiently compared to wild type Ssb5. The RKKR-motif within helix Ssb-αD (Fig. 3a) is prominently exposed when Ssb is bound to ATP5 (PDB 5TKY). It thus appeared reasonably possible that Ssb-ATP was able to interact with ribosomes via the Rpl25 EDD and Ssb RKKR contact. To investigate this possibility, we superimposed Ssb-αD from the ATP-bound structure (PDB 5TKY5) onto Ssb-ADP S1 (Supplementary Fig. 7c). In the resulting Ssb-ATP model, Ssb-SBDβ and Ssb-NBD are detached from the ribosome and the peptide-binding cleft of Ssb-SBDβ is turned away from the tunnel exit (Supplementary Fig. 7c). To test the interaction of Ssb-ATP with ribosomes directly, we investigated whether purified Ssb-ATP interacts with ribosomes, and if this interaction depended on the Rpl25 EDD-motif. To prevent ATP hydrolysis during the experiment, we employed the Ssb-T207A mutant (Supplementary Fig. 7a), which is incapable of hydrolyzing ATP5,39,40. A side-by-side comparison of purified Ssb in the presence of ADP and purified Ssb-T207A in the presence of ATP revealed that the affinity of wild type and mutant for non-translating ribosomes was rather similar (Fig. 3e,f, and see below). Moreover, binding of Ssb-ADP as well as Ssb-T207A-ATP to purified Rpl25-KKK ribosomes was significantly reduced (Fig. 3e,f). We conclude that Ssb-ATP directly interacts with non-translating ribosomes and that this interaction depends crucially on the RKKR-motif within Ssb and the EDD-motif within Rpl25.

Direct ribosome-binding of Ssb-DD1-DD2 was severely impaired (Fig. 3b,c), yet did not affect yeast growth (Supplementary Fig. 7a). It was previously shown that Ssb-ATP can also associate with ribosomes indirectly, through interaction of the Ssb-NBD with the NBD of the RAC subunit Ssz12,28. Mutations in Ssb (R261D/E370R/E547R, termed Ssb-3*), which significantly reduce indirect ribosome association of Ssb via Ssz1, likewise did not affect yeast growth under normal conditions28. We therefore asked whether a combination of the Ssb-DD1-DD2 with Ssb-3* mutations might result in synthetic growth defects. The Ssb-3* mutations caused moderate growth defects in our strain background (Fig. 3g and Supplementary Fig. 7d). Importantly, growth defects were significantly enhanced in a strain expressing Ssb-3*-DD1-DD2, which combines the two sets of mutations (Fig. 3g and Supplementary Fig. 7d). The synthetic growth defect suggested that proper function of the RAC-Ssb cycle was dependent on positioning of Ssb-ATP at the ribosome, either through the interaction of Ssb with Rpl25 or through interaction between the NBDs of Ssb and Ssz1. To test this possibility, ribosome-binding of Ssb-DD1-DD2 was compared with that of the Ssb-3*-DD1-DD2 mutant and with Ssb-DD1-DD2 expressed in the Δssz1 background (Fig. 3h and Supplementary Fig. 7e). According to the dual binding site model, the latter two mutants should exhibit markedly reduced binding. Indeed, ribosome-binding of Ssb-3*-DD1-DD2 and of Ssb-DD1-DD2 expressed in the Δssz1 background was further reduced by more than fivefold (Fig. 3h and Supplementary Fig. 7e) compared to the already strongly reduced ribosome-binding of Ssb-DD1-DD2. These findings demonstrate that residual ribosome-binding observed for Ssb-DD1-DD2 is mediated by the interaction between the Ssb-NBD and Ssz1.

Ssb binds to non-translating ribosomes with high-affinity, which is further enhanced when Ssb interacts with a nascent chain
To quantitatively characterize Ssb-ribosome interactions, we measured binding affinities with ATTO647N-labeled Ssb (Supplementary Fig. 8a,b). Fluorescence anisotropy changes followed a saturation binding curve, reflecting a single binding site with a Kd of 182 ± 50 nM for Ssb in the absence of nucleotide (Fig. 4a). Addition of ADP or of the non-hydrolysable ATP analog AMPPNP resulted in a slight but significant affinity increase (Fig. 4a). We conclude that Ssb-ADP (Kd: 56 ± 19 nM) as well as Ssb-AMPPNP (Kd: 71 ± 8 nM) bind to non-translating ribosomes with similar, submicromolar affinity (Fig. 4a). To address the role of RAC in ribosome-binding of Ssb, we tested whether RAC affected the affinity of Ssb for non-translating ribosomes. To that end, 1:1 ribosome:RAC complexes were generated (Supplementary Fig. 8c), and the binding of Ssb to ribosomes was compared with that to ribosome⋅RAC complexes (Supplementary Fig. 8d-f). The Kd of Ssb for ribosome⋅RAC complexes was 163 ± 68 nM (Supplementary Fig. 8f), which resembled closely the Kd of Ssb for ribosomes (Fig. 4a and Supplementary Fig. 8f). The data suggested that RAC does not enhance the affinity of Ssb for empty ribosomes, indicating that both ATP- and ADP-bound Ssb likely associate with non-translating ribosomes primarily through the interaction of the helix Ssb-αD with Rpl25, rather than through interaction of the NBDs of Ssb and Ssz1 (Fig. 3g,h). However, in cell extract-based systems, ribosome-binding of catalytically inactive Ssb, or wild type Ssb in the absence of RAC, was significantly reduced when compared to wild type Ssb in the presence of RAC (Supplementary Fig. 8g, h and 5).

Since the interaction of Ssb with nascent chains depends on ATP hydrolysis, and RAC stimulates ATP hydrolysis by Ssb11,21, the most plausible explanation for this discrepancy was that the interaction of Ssb with nascent chains established an additional binding site, enhancing the overall affinity of Ssb for ribosomal complexes. To directly test this hypothesis, we analyzed the relative occupancy of Ssb on FLAG-Pgk1-70 RNCs, to which Ssb binds efficiently (Supplementary Fig. 1d). In the presence of RAC, wild type Ssb was bound to RNCs in a salt-resistant manner (Fig. 4b). In the absence of RAC, significantly less Ssb was bound, and Ssb was released from RNCs upon high-salt treatment (Fig. 4b). RNC-binding of the ATPase-deficient Ssb-T207A mutant in the presence of RAC resembled RNC-binding of wild type Ssb without RAC. The data show that ATP-hydrolysis was required for efficient and salt-resistant binding of Ssb to RNCs. We conclude that RAC, by stimulating Ssb´s ATPase activity and allowing Ssb to interact with nascent chains, enhanced the binding affinity and salt-resistance of Ssb.

To understand how these interactions are coordinated structurally, the model of ribosome-bound Ssb in the ATP conformation and the atomic models Ssb-ADP S1 and S2 were overlaid with the atomic model of RAC C2 (PDB 7X3K16,) (Supplementary Fig. 9a–f). Notably, in the model of ATP-bound Ssb, the NBDs of Ssb and Ssz1 were apart, however, faced each other in a way that was compatible with their interaction (Supplementary Fig. 9b, and see Discussion). When Ssb-ADP S1 was overlaid with RAC C2, Ssb-SBDβ was in close proximity of the Ssz1-NBD (Supplementary Fig. 9d), and in the case of Ssb-ADP S2, Ssb-SBDβ overlapped with the Ssz1-NBD atomic model (Supplementary Fig. 9f). These models suggested that RAC can associate with ribosomes bound to Ssb-ATP; however, RAC sterically clashes with Ssb-ADP, particularly in the S2 state. We thus examined whether RAC was bound to FLAG-Pgk1-70 RNCs associated with Ssb-ADP using a two-step purification protocol to isolate RNCs directly associated with Ssb (Fig. 4c). As expected, both Ssb and RAC were present in the initial FLAG affinity-purified RNCs (Fig. 4b and Fig. 4d input). However, following immunoprecipitation of Ssb-bound RNCs, RAC levels fell below the detection limit (Fig. 4d, α-Myc IP). Thus, translating ribosomes carrying a nascent chain bound to Ssb-ADP were no longer stably associated with RAC. We conclude that RAC-mediated ATP hydrolysis by Ssb not only promoted stable engagement with the nascent chain, but also led to RAC release, thereby defining a key mechanistic step in the cotranslational chaperone cycle.

Methods
Media and general growth conditions
Yeast cells were grown at 30°C in YPD medium (1% yeast extract, 2% peptone, 2% glucose, 20µgml-1 adenine hemisulfate) and were harvested in log-phase. To induce translational run-off for the purification of non-translating ribosomes, log-phase cells were transferred to 30°C YP medium (1% yeast extract, 2% peptone) followed by 10 min incubation 30°C and 200 rpm45. E. coli cells for protein expression were grown in LB medium (0.5% yeast extract, 1% peptone, 1% NaCl) on a shaker at 110 rpm at the temperatures detailed below.

Yeast strains
Saccharomyces cerevisiae (S.c.) strain MH272-3f is the parental strain of mutant strains employed in this study46,47. Genomic mutations R596D-K597D-K603D-R604D within SSB1 and SSB2 (Ssb-DD1-DD2 strain) and T207A (Ssb-T207A strain) were generated by CRISPR-Cas9 genome editing48. Gene deletions were generated by genome integration of PCR-amplified DNA fragments containing a selectable marker flanked by 300-500bp up- and down-stream of the target gene. Ssb-T207A and Ssb-DD1-DD2 (with the exception of the strains shown in Fig. 3g,h and related to Supplementary Fig. 7d,e) were integrated into the genome (see below). All other mutated proteins were expressed from plasmids in the respective deletion background. Plasmids, yeast strains, and primer sequences are listed in Supplementary Tables 2-4.

Generation of the genomic Ssb-T207A and Ssb-DD1-DD2 strains
CRISPR-edited clones were selected based on resistance to nourseothricin (pRCC-N) or geneticin (pRCC-K)48. Genomic modifications were verified by sequencing. Loss of pRCC-K and pRCC-N plasmids was by growth of yeast strains on antibiotic-free YPD medium. Primer and single strand donor DNA for CRISPR-Cas9 (dDNA) sequences are listed in Supplementary Table 4. Ssb-T207A strain (Supplementary Table 3) pRCC-K_SSB1/SSB2_PAM634 was constructed by Gibson assembly (GeneArt, Invitrogen)49 of PCR products generated with primer pairs WGP235-pCC2_Fw/CC_Ssb_PAM634 fw and WGP234-pCC1_Rv/CC_Ssb_PAM634 rev and pRCC-K as a template. MH272-3fα was co-transformed with pRCC-K_SSB1/SSB2_PAM634 and dDNA_Ssb_T207A_HindIII. Yeast clones carrying the T207A mutation in SSB1 and SSB2 (Ssb1-T207A and Ssb2-T207A) were selected based on geneticin resistance and slow growth displayed by strains expressing Ssb1-T207A and Ssb2-T207A (Supplementary Fig. 7a). Due to sequence variation in the 3´-region of the SSB1 and SSB2 orfs, the R596D-K597D (DD1) mutation was introduced into SSB1 and SSB2 successively. First, the DD1 mutation was introduced into SSB2. To that end, pRCC-N_SSB2_PAM1789 was generated with primer pair WGP235-pCC2_Fw/CC_Ssb2PAM1789 fwd and WGP234-pCC1_Rv/CC_SSB2PAM1789 rev using pRCC-N as a template. pRCC-N_SSB2_PAM1789 was co-transformed with dDNA Ssb2_DD1 resulting in the strain Ssb2-DD1. Second, the DD1 mutation was introduced into SSB1 using pRCC-K SSB1_PAM1789 generated with primer pair WGP235-pCC2_Fw/CC_Ssb1 PAM 1789 fwd and WGP234-pCC1_Rv/CC_Ssb1 PAM 1789 rev using pRCC-K as a template. pRCC-K SSB1_PAM1789 was co-transformed with dDNA Ssb1/Ssb2_DD1 into Ssb2-DD1, resulting in strain Ssb-DD1. The K603D-R604D (DD2) mutation was then introduced into SSB1-DD1 and SSB2-DD1. To that end, pRCC-K_SSB1/SSB2_PAM1823 was generated with primer pair WGP235-pCC2_Fw/CC_Ssb1/2-PAM1823 fwd and WGP234-pCC1_Rv/CC_Ssb1/2 PAM 1823 rev using pRCC-K as a template. pRCC-K_SSB1/SSB2_PAM1823 was co-transformed with dDNA SSB1-2_DD1_DD2_AatII into Ssb-DD1. The resulting strain, termed Ssb-DD1-DD2, carries the R596D-K597D-K603D-R604D quadruple mutation in SSB1 and SSB2.

Plasmid construction
Primer and synthetic DNA sequences (Eurofins Genomics) for the construction of yeast vectors50 are listed in Supplementary Table 4. pYEplac112-Rpl25 was constructed by transfer of RPL25±300 (primer pair Rpl25-300-PstI-F/Rpl25-300-BamHI-R) into pYEplac11250. pYEplac112-Rpl25-AAA and pYEplac112-Rpl25-KKK were generated by replacing the StyI/PacI fragment within pYEplac112-Rpl25 with synthetic DNA encoding for the corresponding StyI/PacI fragments encoding for RPL25-E77A-D131A-D134A or RPL25-E77K-D131K-D134K, respectively (Supplementary Table 4). For the construction of Ssb1 mutants SSB1±300bp (primer pair Ssb1-BamHI-F/Ssb1-PstI-R) was introduced into pSP65 (Promega). For the generation of the single cysteine mutant, the ClaI/HindIII fragment within SSB1 was replaced with a synthetic DNA fragment encoding for SSB1-C454S-C435S-C20S (Ssb1-C454S-C435S-C20S, Supplementary Table 4) resulting in pSP65-Ssb1-C454S-C435S-C20S. The HindIII/XbaI DNA fragment within the SSB1-C454S-C435S-C20S DNA sequence was then replaced with a synthetic DNA fragment encoding for the D589C mutation (Ssb1-D589C, Supplementary Table 4), resulting in pSP65-Ssb1-C454S-C435S-C20S-D589C. SSB1-C454S-C435S-C20S±300bp and SSB1-C454S-C435S-C20S-D589C±300bp were transferred into the BamH1/Pst1 site of pYCplac33, resulting in pYCplac33-Ssb1-C3S and Ssb1-C3S-D589C, respectively, which were expressed in the Δssb1Δssb2 background (Supplementary Table 2). The resulting Ssb1-C3S and Ssb1-C3S-D589C strains display wild type growth (Supplementary Fig. 8a). pET28a(Novagen) was employed for the expression of N-terminally His6-tagged wild type, or mutant versions of Ssb1 in E. coli. For that purpose, SSB1-T207A was amplified from Ssb-T207A genomic DNA, SSB1-DD1-DD2 from Ssb-DD1-DD2 genomic DNA, and SSB1-C3S-D589C from pYCplac33-Ssb1-C3S-D589C with primer pair Ssb1-NdeI-F1/Ssb1-BamHI-R1. Each PCR product was cloned into pET28a. The resulting plasmids are termed pET28a-His6-Ssb1-T207A, pET28a-His6-Ssb1-DD1-DD2, and pET28a-His6-Ssb1-C3C-D589 (Supplementary Table 1). For the expression of RAC, the SSZ1 orf was transferred into the NdeI/XhoI site of pET28N18 resulting in pET28N-His6-Ssz1, which was co-transformed with pETcoco2-Zuo118. Dap2±300bp carrying a C-terminal 3x HA-tag was cloned into the PstI/BamHI site of pYEplac195, resulting in pYEplac195-Dap2-3HA. Low copy plasmids for the expression of Ssb1 mutants in the Δssb1Δssb2 background are based on pYCplac-33-Ssb118. pYCPlac33-Ssb1-3* and pYCPlac33-Ssb1-3*-DD1-DD2 were generated by replacing the AleI/PstI fragment of pYCplac-33-Ssb1 with synthetic DNA Ssb1-R261D-E370R-E547R (Supplementary Table 4) or Ssb1-R261D-E370R-E547R-R596D-K597D-K603D-R604D (Supplementary Table 4), respectively. In addition to the mutations within Ssb1, these synthetic DNAs introduced a PacI site 3´ of the Ssb1 stop codon (Supplementary Table 4). pYCPlac33-Ssb1-DD1-DD2 was constructed by replacing the AleI/PacI fragment of pYCPlac33-Ssb1-3*-DD1-DD2 with a PCR product generated with the primer pair Ssb1-AleI-F/Ssb1-PacI-R (Supplementary Table 4) using pET28a-His6-Ssb1-DD1-DD2 as a template. pYCPlac33-Ssb1-DD1-DD1, pYCPlac33-Ssb1-3*, and pYCPlac33-Ssb1-3*-DD1-DD2 were transformed into Δssb1Δssb251 and pYCPlac33-Ssb1-DD1-DD2 was transformed into Δssb1Δssb2Δssz1. The resulting strains are termed Ssb1-DD1-DD2, Ssb1-3*-DD1-DD2, and Δssz1 Ssb1-DD1-DD2.

In vitro transcription and translation
DNA templates for transcription reactions were PCR-amplified using pSPUTK-Dap2, pSPUTK-Pgk1 or pSPUTK-FLAG-Pgk137 as a template. Reverse primers (Supplementary Table 4) were designed such that PCR products encoded stop codon-less FLAG-Pgk1-40, FLAG-Pgk1-50, FLAG-Pgk1-60, FLAG-Pgk1-70, FLAG-Pgk1-80, FLAG-Pgk1-87, FLAG-Pgk1-100, Pgk1-120, and Dap2-120, respectively. Pgk1 and Dap2 transcripts were generated using SP6 polymerase (Thermo Fisher Scientific)52. Yeast translation extracts were prepared from wild type, Δssb1Δssb2, Δssb1Δssb2Δzuo1Δssz1, and Δsrp5452. Ribosome nascent chain complexes (RNCs) were generated using yeast translation extracts52 primed with stop codon-less FLAG-Pgk1, Pgk1, or Dap2 transcripts of the length indicated in the Figure Legends21,53. Translation reactions were performed with 35% yeast translation extract in translation buffer (1.0 mM ATP, 0.08 mM GTP, 17.5 mM creatine phosphate, 0.2 mg/ml creatine phosphokinase, 0.2 mM of each amino acid, 2.2 mM putrescine, 0.1 mg/ml yeast tRNA, 100 U/ml RiboLock (Thermo Fisher Scientific)) at 20°C as previously described53,54. After 80 min incubation at 20°C translation reactions were stopped by addition of cycloheximide (final concentration of 50 µg ml-1). Ribosomal proteins, ribosome-associated factors, and FLAG-tagged nascent chains were detected by immunoblotting using the antibodies indicated in the Figures. In some experiments, RNCs were generated in the presence of [³⁵S]-methionine (Hartmann-Analytic) to enable radiolabel-based visualization of the nascent chain. In these experiments unlabeled methionine was excluded from the translation reaction.

Chemical crosslinking and identification of crosslink products by denaturing immunoprecipitation
RNCs carrying nascent Dap2-120 or Pgk1-120 were generated as described above, were isolated by centrifugation, and were resuspended in ribosome-binding buffer (20 mM HEPES-KOH, 2 mM Mg(OAc), 120 mM KOAc, 50 μg ml-1 cycloheximide, 2 mM dithiothreitol, 1 mM PMSF (phenylmethylsulfonylfluoride), 1x PIM (protease inhibitor mix), pH 7.4). Crosslinking was performed by addition of the amino-reactive, homo-bifunctional crosslinker bis-(sulfosuccinimidyl)-suberate (BS3, Thermo Scientific), which was added to a final concentration of 400 μM. Immunoprecipitation with crosslinked samples was performed under denaturing conditions using protein A-Sepharose beads (CL-4B; GE Healthcare) pre-coated with α-Ssb37.

FLAG- and Myc-affinity purification of RNCs
For FLAG-affinity purification (Fig. 4b), RNCs containing FLAG-tagged Pgk1-70 were generated in translation extracts derived from either Δssb1Δssb2 or Δssb1Δssb2Δzuo1Δssz1 strains. Translation reactions were layered onto either low-salt (25% w/v sucrose in ribosome-binding buffer) or high-salt (25% w/v sucrose in ribosome-binding buffer supplemented with 800 mM KOAc) sucrose cushions and were centrifuged at 400,000 xg for 35 minutes at 4°C. Ribosomal pellets containing RNCs were resuspended in IP buffer (20 mM HEPES-KOH, 2 mM Mg(OAc), 120 mM KOAc, 2 mM dithiothreitol, 50 µg ml-1 cycloheximide, 1 mM PMSF, 1x PIM, pH 7.4). Where indicated (Fig. 4b), purified His₆-Ssb or His₆-Ssb-T207A was added to a final concentration of 1 µM, followed by FLAG-affinity purification using anti-FLAG M2 affinity gel (Sigma)55. For the two-step purification (Fig. 4c), FLAG-Pgk1-70 mRNA was translated in Δssb1Δssb2 extract for 80 minutes at 20°C. Translation reactions were then supplemented with ribosome-free total cell extract from either mycSsb1-expressing cells or wild type (control), prepared by centrifugation of total extract at 400,000 xg for 35 minutes. After a 10-minute incubation at 20°C, FLAG-Pgk1-70 RNCs were isolated using FLAG-beads. Subsequently, FLAG-beads were washed with IP buffer and FLAG-Pgk1-70 RNCs were eluted with FLAG peptide (200 µg ml⁻¹ in IP buffer). To isolate FLAG-Pgk1-70 RNCs associated with mycSsb1, eluted FLAG-Pgk1-70 RNCs were subjected to a second affinity purification using anti-Myc agarose beads (Thermo Scientific). After washing with IP buffer, Myc-beads were boiled in SDS sample buffer and were analyzed by immunoblotting with the antibodies indicated in Fig. 4b,d.

Purification of RNCs and preparation of cryo-EM grids
RNCs carrying FLAG-Pgk1-70 were generated in Δssb1Δssb2 extract complemented with purified, untagged Ssb1, or complemented with untagged Ssb and His6-Snl1-ΔTM (Supplementary Fig. 6f) each at a final concentration of 3 µM). His6-Snl1-ΔTM was included in the translation reaction because it was previously suggested that the BAG-domain protein Snl1, lacking its single transmembrane domain (Snl1-ΔTM) facilitates the recruitment of Ssb to translating ribosomes56. For the purification of FLAG-Pgk1-70 RNCs for cryo-EM analysis, translation reactions were diluted 1:5 with IP-buffer, RNCs carrying FLAG-Pgk1-70 were affinity purified using FLAG-beads, and were subsequently released with FLAG-peptide (see above and 55). Four µl of released RNCs FLAG-Pgk1-70 (RNC concentration ~50 nM) were incubated on R3.5/1 200 mesh copper grids (Quantifoil) prefloated with 2nm carbon for 30s, were blotted for 1s, and were subsequently plunge frozen in liquid ethane using an EM-GP2 plunge-freezer (Leica). The temperature in the loading chamber was set to 4°C and the humidity in the loading chamber was set to 75%.

Data collection and image processing
Cryo-EM data were collected on grids with FLAG-Pgk1-70 RNCs prepared in the presence of Ssb, with or without His6-Snl1-ΔTM. Initial reconstructions revealed Ssb density near the tunnel exit in both samples, while no density for His6-Snl1-ΔTM was observed. The sample containing His6-Snl1-ΔTM showed more clearly defined Ssb density, which could potentially indicate a positive effect of Snl1-ΔTM on Ssb recruitment or positioning at the tunnel exit. However, we consider it more likely that this variation reflects the intrinsic variability of cryo-EM data obtained from affinity purified, in vitro generated RNCs, as well as differences in cryo-EM sample preparation, grid handling, and data collection. As more defined Ssb density provided an advantage for subsequent data collection, the His6-Snl1-ΔTM dataset was selected for further analysis. Movies were collected using a 300 kV Titan Krios G4 equipped with a cold field emission gun, a Selectris energy filter (Thermo Fisher Scientific) with 10 eV slit width and a Falcon 4i direct electron detector. Micrographs were acquired at 130,000x magnification (pixel size 0.951 Å/px) and a cumulative fluence of 40 e/Å2 using a defocus range between -1.0 to -2.0 µm in 0.25 µm increments in EER format (40x fractionated)57. Data collection was automated using EPU (Thermo Fisher Scientific). The 9995, 10476 and 36270 movies were patch motion corrected and CTF estimated in CryoSPARC live (v4.2.1, 4.4.1 and 4.5.1) (v4.2.1, 4.4.1 and 4.5.1)58. Defocus and CTF fit values were used to curate the exposures to 9606, 10471 and 34209 micrographs, respectively. Particle picking was performed using cryolo (v 1.9)59 by refining pretrained weights using 10 to 15 manually picked micrographs. Extracted particles (180 px 2.64 Å/px) were separated into batches of around 225,000 particles and sorted in four rounds of 2D classifications using CryoSPARC (v. 4.2.1 to 4.5.1). Ab initio reconstruction was performed on the first collection to obtain 80S and 60S reference volumes that were used for particle sorting in heterogeneous refinements. 80S particles were refined and used for cryoDRGN variational autoencoder training (128 px, 3.71 Å/px, 3 layers for encoder and decoder each, for 50 epochs in an 8-dimensional latent space)60. Using the cryoDRGN landscape’ tool, latent space encodings were first clustered via kmeans (k=1000) and the resulting reconstructions, generated by backprojection, were further clustered via agglomerative clustering (k=20) (https://ez-lab.gitbook.io/cryodrgn/). The volumes of the resulting clusters are shown as centroids, representing voxel-based averages of the reconstructions within each cluster (Supplementary Fig. 3a, b). Latent space dimensionality reduction was performed by uniform manifold approximate and projection (UMAP). The resulting reconstructions of ribosomal states across the translation cycle were analyzed for density near the ribosomal tunnel exit, the hypothesized binding site of Ssb. Best resolved density was observed in the cluster representing non-rotated, post-translocation state ribosomes, with a P-site tRNA and a peripheral density corresponding to a trailing ribosome (Supplementary Fig. 3, Custer 1). Corresponding particles were re-extracted (180 px box size, 2.64 Å/px), converted to relion star files using cs2star/pyem, (https://github.com/brisvag/cs2star)61. The particle stacks from the combined collections 1 and 2 as well as collection 3 were refined in relion (v 5.0)62 followed by a focused 3D classification (10 classes, T=300) masking around the Ssb density. The ellipsoid mask was generated in ChimeraX (v. 1.7) using the ‘shape’ command followed by the ‘volume ones mask’ command. Subsequently, the volume was binarized and dilated by a ‘soft-edge’ (2 px, 2.64 Å/px) in CryoSPARC58. The particles from collection 1+2 were combined with particles from the collection 3 for each Ssb conformation (S1 and S2) followed by further 3D classification masking around the Ssb density in both states by using the eraser sphere tool followed by binarization and dilation in CryoSPARC58. Particles were refined and 3D classified (2 classes, target resolution 6 Å, forced hard classification) once more in CryoSPARC using previously generated masks, followed by another round of refinement and 3D classification focusing on the P-site tRNA using a mask generated with the volume eraser tool as previously described. Particles with more defined tRNA density were selected for homogeneous refinement. The resulting maps were post processed via DeepEMhancer using the ‘high-res’ weights provided by the developers63 resulting in cryo-EM maps Ssb-ADP S1 and S2 (Fig. 1b). Local resolution estimation was performed with CryoSPARC58.

Model building
A non-rotated, post-translocation ribosome with P- and E-site tRNA PDB 6T7I64 was fitted in our densities as a rigid body fit in chimera (v 1.7-1.865) as an initial model, together with the AlphaFold2 prediction66 of the Ssb-SBD. The tRNA was replaced by tRNAMet-e. Next, models were refined manually using coot (v 0.9.867) and Isolde (v 1.868). The nascent chain was modeled de novo using coot. Subsequently, real space refinement was performed using phenix (v 1.2169,70), followed by assessment of model statistics by phenix validation71,72. Problematic regions were manually fixed and the model was refined in phenix iteratively.

Structure prediction
The structures of full-length S.c. Ssb-ATP and S.c. Ssb-ADP were predicted by I-TASSER73 using Chaetomium thermophilum (C.t.) Ssb-ATP (PDB 5TKY5) and E. coli DnaK-ADP (PDB 2KHO29) as a template. The complex consisting of Ssb-ATP, Zuo1, and Ssz1 was predicted by AF374 (Supplementary Fig. 10 and Supplementary Note 2). Generation of models for the RAC-Ssb chaperone cycle (Fig. 5) is detailed in Supplementary Note 2 and Supplementary Fig. 10. Animated models (Supplementary Movies 1-5) were created using the morph function in ChimeraX (1.8-1.9) and structures presented in Fig. 5. To avoid clashes, morphing was performed using manually created intermediate structures.

Heterologous protein expression and purification
E. coli expression plasmids are listed in Supplementary Table 1. His6-Ssb1, His6-Ssb1-T207A, His6-Ssb1-DD1-DD2, His6-Ssb1-D589C, and His6-Snl1-ΔTM were expressed in Rosetta (DE3) (Novagen). For the purification of RAC, His6-Ssz1 and Zuo1 were co-expressed in E. coli BL21 (DE3). Expression was induced with 1 mM isopropyl-β-D-1-thiogalactopyranoside at an OD600 of 0.5 followed by 4 hours incubation at 25°C for the expression of His6-Ssb1 and His6-Ssb1-variants, 4 hours incubation at 26°C for expression of RAC, and for 4 hours incubation at 37°C for expression of His6-Snl1-ΔTM. His-tagged proteins were purified using ÄKTA start (GE Healthcare) and a Ni²⁺-NTA column (PureCube Ni-NTA Cartridge 5 ml, Cube Biotech). Elution of proteins bound to the Ni²⁺-NTA column was with a 30 ml linear 50-500 mM imidazole gradient in NiP1-buffer (20 mM HEPES-KOH, 100 mM KCl, 2.5 mM Mg(OAc₂), 1 mM PMSF, pH 7.8). RAC was purified by Ni²⁺-NTA purification followed by a second purification step using anion-exchange chromatography on MonoQ-5/50 GL (GE Healthcare) with a 20 ml linear 50-500 mM NaCl gradient in 20 mM HEPES-KOH, pH 7.4. His6-Snl1-ΔTM was purified by Ni²⁺-purification followed by size-exclusion chromatography (ÄKTApure, GE Healthcare) on Superdex 200 10/300 GL (GE Healthcare) equilibrated with NiP1-buffer adjusted to pH 7.4. Eluted proteins were transferred to NiP2 buffer (20 mM HEPES-KOH, 100 mM KCl, 2.5 mM Mg(OAc)2, pH 7.4) with PD10 columns (GE Healthcare). Purified His6-Ssb1 variants and RAC preparations are shown in Supplementary Fig. 6f. For the preparation of cryo-EM samples the His6-tag was removed from purified His6-Ssb1 by cleavage with thrombin (Thrombin Clean Cleave, Sigma) in cleavage buffer (20 mM HEPES-KOH, 100 mM KCl, 2.5 mM Mg(OAc)2, pH 7.8) at 20°C overnight. After addition of 2 mM PMSF, thrombin agarose beads were removed by centrifugation at 500 xg and the supernatant containing untagged Ssb was clarified by centrifugation at 20,000 xg for 20 min at 4°C, was transferred to NiP2 buffer containing 1x PIM as described above (Supplementary Fig. 6f).

Purification of non-translating ribosomes
Ribosome-binding assays and Anisotropy experiments were performed with freshly prepared non-translating ribosomes. Example blots are shown in Supplementary Fig. 6d,e. Ssb-free non-translating ribosomes were obtained from the mycSsb1-ΔC23 strain5, which does not suffer from ribosome assembly defects observed in Δssb1Δssb2 strains54. Ribosomes carrying Rpl25-E77K-D131K-D134K were purified from the Rpl25-KKK strain. Typically, a 2l log-phase YPD culture was glucose-depleted as described above. After harvest, glucose-depleted cells were rapidly frozen in liquid nitrogen, and subsequently disrupted with a cryo-mill (MM400, Retsch). The cryo-mill powder was resuspended in high-salt lysis buffer (20 mM HEPES-KOH, 100 mM KOAc, 500 mM KCl, 2.5 mM Mg(OAc)2, 1x PIM, 1 mM PMSF, 1 mg ml-1 Heparin, 2 mM dithiothreitol, pH 7.4) at a ratio of 1 mg cryo-mill powder per 5 ml buffer. The resulting lysate was clarified by centrifugation at 20,000 xg for 10 min at 4°C followed by a second centrifugation step at 40,000 xg for 20 min at 4°C. Fifteen to 20 ml of the clarified supernatant was loaded onto a 2.5 ml high-salt sucrose cushion (25% sucrose (w/v) in high-salt lysis buffer) and ribosomes were collected by centrifugation at 400,000 xg at 4°C in T865 rotor (Thermo Scientific). Ribosomes were resuspended in high-salt lysis buffer and were incubated for 30 min at 4°C and 1000 rpm (IKA VXR Basic Vibrax Shaker) followed by a 2 min clarifying spin at 2,000 xg. Solubilized ribosomes (500 µl) were loaded onto a 500 μl high-salt sucrose cushion and ribosomes were collected by centrifugation for 30 min at 400,000 xg in a MLA-130 rotor (Beckman Coulter) at 4°C. Ribosomes were resuspended in storage buffer (20 mM HEPES-KOH, 100 mM KCl, 2.5 mM Mg(OAc)2, pH 7.4) and were incubated at 1,000 rpm 4°C for 1 h followed by a 2 min clarifying spin at 2,000 xg 4°C prior to use.

Ribosome-binding assays
Ribosome-binding assays with total cell extract were performed with cycloheximide (final concentration 100 µg ml-1) treated log-phase cells. Cells were resuspended in ribosome-binding buffer and were disrupted by the glass beads method5. After a clearing spin at 20,000 xg, each 60 µl of the total cell extract (A260 between 90 and 100 mAu) was loaded onto a 90 µl low-salt sucrose cushion (25% (w/v) sucrose in ribosome-binding buffer or onto a 90 µl high-salt sucrose cushion (25% (w/v) sucrose in 20 mM HEPES-KOH, 800 mM KOAc, 2 mM Mg(OAc)2, 50 μg ml-1 cycloheximide, 2 mM dithiothreitol, 1 mM PMSF, 1x PIM, pH 7.4). After centrifugation at 400,000 xg for 35 min at 4°C total cell extract, cytosolic supernatant and ribosomal pellets were analyzed as described5. For ribosome-binding assays with purified components typically 2 µM non-translating ribosomes were incubated with 1 µM His6-Ssb1 or His6-Ssb1-variants in ribosome-binding buffer lacking cycloheximide for 2 min at 25°C. After a clarifying spin at 2000 xg, 20 µl of the sample was loaded onto a 30 µl low-salt sucrose cushion. Ribosomes and bound Ssb were isolated by centrifugation at 400,000 xg (TLA100, Beckman Coulter) at 4°C. Ribosome-binding assays in the presence of nucleotide were performed with purified His6-Ssb or His6-Ssb variants pre-incubated at 22°C with 1 mM ADP or ATP for 2 min in ribosome-binding buffer containing 1 mM ATP or ADP as indicated.

Anisotropy measurements
Purified His6-Ssb1-C3S-D589C (100-200 µM) was incubated with a two-fold molar excess of Atto647N maleimide (ATTO-TEC) in NiP2 buffer with 1x PIM. The reaction was incubated overnight at 20°C and was then quenched with dithiothreitol (1 mM final concentration). Labeling efficiency, determined by absorption spectroscopy (NanoDrop 2000, Peqlab) according to the ATTO-TEC protocol, was above 88% in all preparations (Supplementary Fig. 8b). Free dye was removed by gravity flow using PDG-25 MiniTrap columns (Cytiva) equilibrated with NiP2 buffer. Anisotropy measurements were performed in storage buffer using a plate reader (TECAN Spark, 28°C, λex = 635 nm, λem = 680 nm, 20 nm excitation and emission slit). The concentration of His6-Ssb1-ATTO647N was held constant (100 nM) and increasing concentrations of non-translating ribosomes or 1:1 ribosome⋅RAC complexes (Supplementary Fig. 8c) was added. Ssb was pre-incubated with 1 mM ADP or AMPPNP (Sigma) as indicated prior to addition of ribosomes in NiP2 buffer, containing 1 mM ADP or AMPPNP, respectively. To determine dissociation constants, Anisotropy values were plotted as a function of increasing ribosome concentrations and were fitted to a 1:1 binding model with GraphPad Prism 10.1.1 using equation
r = r_unbound + (r_bound - r_unbound) * [SR] / [S0]
where [SR] was replaced by
[SR] = (S0 + R0 + Kd - sqrt((S0 + R0 + Kd)^2 - 4*S0*R0)) / 2
Anisotropy values were weighted by 1 over their variance. Eight biological replicates were performed to determine Kd values of Ssb for non-translating ribosomes in the absence of nucleotide, 3 biological replicates were performed to determine Kd value of Ssb for the ribosome⋅RAC complex, and 3 biological replicates were performed to determine Kd values of Ssb for non-translating ribosomes in the presence of ADP or AMPPNP, respectively.

Quantification and statistical analysis
Quantitative analysis of immunoblots was with ImageJ 1.54g (National Institutes of Health, USA). Statistical analysis was performed with at least 3 biological replicates using GraphPad Prism 10.1.1. The number of replicates used for statistical analysis is indicated in the Figure Legends. Analysis of Variance (ANOVA) with Tukey´s test for multiple comparison was used to determine statistic significance. Statistical analysis for ribosome-binding assays was by Two-way ANOVA, with the exception of Supplementary Fig. 8e (paired t-test). p values are indicated in the Figures. p values > 0.05 are regarded as nonsignificant.

Antibodies
Rabbit polyclonal antibodies α-Ssb21 (1:10,000, #471), α-Zuo153 (1:20,000, #10), α-Ssz153 (1:8,000, #11), α-Rpl355 (1:5,000, #99), α-Srp5453 (1:2,000, #12), α-Sse153 (1:10,000, #31), α-Rpl2555 (1:5,000, #101), α-Rpl475 (1:8,000, #167), α-Rps953 (1:5,000, #44), and α-Pgk175 (1:10,000, #407) (Eurogentec, Rospert lab antibody collection). α-His6 (1:4,000, Biorad, 154295), α-FLAG-tag (1:2,000, Sigma, F1804), α-HA (1:2000, Invitrogen, 26183) are purified monoclonal antibodies. Horseradish peroxidase-conjugated protein A (1:5,000, Thermo Fisher Scientific, 101023) or goat anti Mouse HRP (1:10,000, Santa Cruz, sc-2748) was employed as a secondary antibody. Numbers in brackets indicate the antibody dilution for immunoblotting and the antibody collection number).

Miscellaneous
PIM (proteinase inhibitor mix), 1000-fold concentrated in dimethyl sulfoxide: 1.25 mg ml-1 leupeptin, 0.75 mg ml-1 antipain, 0.25 mg ml-1 chymostatin, 0.25 mg ml-1 elastinal, 5 mg ml-1 pepstatin A. For the determination of protein levels in yeast cells, lysates were generated under denaturing conditions76. Protein samples were separated on 10% Tris-Tricine gels77. Immunoblotting of protein samples was onto PVDF (Amersham) or nitrocellulose (Amersham) membrane. Immunoblots were developed by enhanced chemiluminescence using an ECL camera78. Protein concentrations were determined with the Bradford assay79, and in the case of recombinant Ssb1 with the calculated extinction coefficient at 280 nm (εSsb1 = 18910 M−1 cm−1). The concentration of purified ribosomes was determined using the extinction coefficient at 260 nm (εribosome = 6 × 107 M-1 cm-1)80. Autoradiographs and fluorescently labeled proteins were analyzed with Typhoon 9410 (GE Healthcare).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Zhang, Grundmann et al. presents cryo-EM structures of the yeast Hsp70 homolog Ssb bound to the\nribosome. This fills a distinct knowledge gap in the field regarding how this protein folding chaperone can cotranslationally\nengage with substrates. The single particle cryo-EM work is well done, and the interactions visualized in the structures are\nnicely validated with biochemical assays and careful mutant analyses. This work also convincingly demonstrates an impact\nof nascent protein engagement on the salt-resistant binding stability of Ssb to ribosomes. Overall, these results reflect\ncareful, high quality experimentation. However, I found the final mechanistic model to be confusing and unnecessarily\ncomplicated. I believe the manuscript would be improved if the authors did not attempt to seemingly incorporate every single\nobservation related to RAC and Ssb into a comprehensive model and instead focused on emphasizing functional concepts\nthat are clearly demonstrated versus those that may be consistent with alternative interpretations.\nSpecific comments:\n1. Figure 5 presents a mechanistic model with incredibly detailed but incompletely supported steps, in an apparent attempt\nto explain multiple conformational states of RAC bound to the ribosome (4 states), and separately, of Ssb bound to the\nribosome (2 states). Although I appreciate the effort put into reconciling all these states, it does not seem necessary, and\nmany of the steps appear to be conjecture. In my opinion, these rather confusing and detailed analyses detracts from the\nhigh-quality work in this study. I would favor removing most of these descriptions and significantly simplifying the Discussion\nfor several reasons:\na. The RAC conformational states were reported in previous studies, and there is not enough space to clearly explain what\nthe four different states are. As a result, the Discussion covering these states is incredibly dense with overly specialized\nnomenclature.\nb. It is not clear to me why all states of RAC and Ssb observed by cryo-EM must be considered functionally relevant or part\nof the same mechanistic pathway. There are numerous examples of parallel factor-ribosome engagement pathways. Cryo-\nEM also may capture states that are artificially stabilized during lysis and isolation procedures. It does not seem like there is\nsufficient evidence to suggest that all of the observed states must be put into the same pathway, that they are as precisely\ncoordinated as indicated in Figure 5, or that Ssb loading may not occur slightly differently for different ribosome-\nnascent protein complexes.\n2. The nascent protein density outside of the ribosomal exit tunnel is not clear in the provided maps. Is it only visible in\nunsharpened maps? The maps provided also appear to have gone through a sharpening procedure, but sharpening B-\nfactors are not provided in the statistics table.\n3. How was ADP assigned to the ribosome-bound Ssb in the structure? Was this by inference or by structural modeling?\nThis reasoning could be clearer.", "reviewer_2": "In the manuscript \"The cotranslational cycle of the ribosome-bound Hsp70 homolog Ssb\", Ying Zhang and coworkers\npresent a structure of the Hsp70 chaperone, Ssb, bound to the yeast ribosome. They observe good density for the SBDa\ndomain, which allows them to localise a contact with Rpl25. This interaction interface is then tested with a series of\nexperiments involving mutagenesis and binding assays. Finally, the authors combine this new structure with the previous\nstructure of RAC to provide a theoretical model for the cotranslational protein biogenesis process. The structure of ribosome-\nbound Ssb is a significant result as it is one of the best (higher resolution) structures reported to date, to my knowledge, for\nthis important system.\nFrom the cryoEM perspective, the presented structures support the main conclusion regarding the interaction interface.\nSpecifically, the cryoEM map in this region is of good quality, allowing the authors to place SDBDa confidently and to\ndescribe the potential interaction interface between the SBDa domain and Rpl25. Namely, a complementary electrostatic\npatch is identified on Ssb and Rpl25. The mutagenesis and binding experiments that validate this potential electrostatic\ninteraction appear to be well-performed and logical.\nSpecific Comments:\n1) In the main text, the nature of the studied complex is not well explained and has to be understood from the Methods\nsection. For example, it could be clearer that the complex is prepared in vitro by mixing purified protein with RNCs made\nfrom an in vitro translation reaction (as opposed to isolating an in vivo assembled complex). The methods also mention that\na protein, His6-Snl1-∆TM, was also added, but it is not well explained what its role is, and if it is observed in the structure.\n2) It would be useful to make it clearer to the reader exactly which parts of Ssb's structure were solved in relation to the\nribosome. This could be facilitated by including the domain structure drawing of Ssb (Extended Data Figure 1B) as part of\nFigure 1 and also indicating in the text which residues are truly solved at high resolution, which are primarily docked from a\nmodel and which are disordered. In line with this, a close-up view of the SSB density (i.e., using a volume zone in ChimeraX\nor a segmented map), coloured according to local resolution, with the model inside, would help convey the confidence in the\nSBDa model, which forms the binding interface. Similarly, the lower local resolution in SBDb could indicate inherent\nflexibility in SBDb, which could be important for function.\n3) The text indicates that Ssb is in the ADP form, but it is unclear if ADP or ATP is in the buffer or co-purifies with the protein.\nI guess the ADP form is inferred from the domain arrangement of Ssb, but this could be clarified in the text.\n4) Neither of the figures shows density for all 17 aa of the NC. A more complete figure would help the reader understand if\none can assign sidechains for all 17 (line 64) or only bulky residues (i.e. Tyr).\n5) In Figure 2A, the nature of the map shown is unclear. Is it unsharpened, sharpened or low-pass filtered? The sharpened\nmap provided appears to show more details than this figure. In the main text, it could be made more explicit that Figure 2A\nrefers to State S1 and the extended data Figure 5A to State S2, as the figures are very similar on first glance. The interaction\ncould be described better as some of the mentioned residues are positioned to interact directly, while others are more\nindirect.\n6) Line 653: It could be useful to explain how the mask was generated.\n7) Line 644: More details could be provided for the training, for example, the encoder/decoder dimensions and number of\nlayers, number of epochs, and whether filtering was done. In the cryodrgon step, a lot of particle projections are eliminated.\nAre these the rotated ribosomes? Does the rotation state have a substantial effect on Ssb binding, and if so, why would this\nbe? Would it be possible to combine rotated and non-rotated particles by using a refinement focused on the large subunit (or\nmaybe using particle subtraction, multibody refinement)? A larger dataset could improve the density. Has this been tried to\nno effect?\n8) Figure 1D The rendering of the density in the figure does not really give it a 3D feel to judge if density for the NC is\npresent or not. Moreover, the low local resolution of SBDb domain, in general, could make it challenging to say the NC is\nthere confidently (i.e. line 63-64). That said, there is an extension (colored in red in Figure 1D) that leads out of the\nSBDb domain towards the tunnel exit. Perhaps the text \"clear nascent chain density in both states\" could be rephrased as follows:\n\"There is an extension (red density, Figure 1D) leading away from the SBDb density towards the NC tunnel exit, which we\ntentatively attribute to the NC.\"\n9) In the local resolution panels, does red indicate 6.9 Å or does it indicate > 6.9 Å? I mean, for example, when colouring the\nmap, is the range set to a max of 6.9, so any voxels with a value of 6.9 or above are rendered in red? What package was\nused for the local resolution calculation?\nMinor Comments:\n1) The company Thermo Fisher Scientific is referred to as both \"ThermoFischer Scientific\" and \"Thermo Scientific\" in the\nmethods. It should be consistent and spelt correctly\n2) Line 632, the provided magnification is not consistent with Table 2\n3) Table 1 indicates the FSC threshold is 0.5, but in the Extended Data Figure 2B and E, it appears that 0.143 is used\n4) Line 807: In \"EMPIAR-XYZ\", XYZ can be updated.\n5) Extended Data Fig. 2. The number of particles in the lower part of the State S1 side should be double-checked. The figure\nindicates that 1.28x10^5 particles go into the 3D classification, then 53% are selected. In the next step after the selection, the\nparticles have increased to 1.31x10^5 and again increase after the next classification to 6.14x10^5.\n6) Extended Data Figure 5 in some binding assays, the fractions are designated T, C, and R and in other T, S, and R (i.e.\nExtended Data Figure 7). The reason for the difference is unclear.\n7) Extended Data Figure 5D. Is it possible the western blots have been shifted up, as here Ssb and rpl4 run at 116 and 66\nkDa, while in Extended Data Figure 5G they run at 66 and 35 kDa\n8) Extended Data Figure 4 right-hand panels. It would be useful to have the L25 label.", "reviewer_3": "Co-translational folding of proteins by chaperones is a critical process in protein homeostasis. Hsp70s are an abundant\nclass of chaperones that interact with and facilitate folding of nascent chains. In yeast, the Hsp70 Ssb has a cochaperone,\nRAC, comprising Zuo1 and SSz1. How Ssb and RAC function together on the ribosome is not well understood. In this\nmanuscript, the authors use single particle cryo-EM as well as biochemical and genetic approaches to examine this\nquestion. The principal finding of the manuscript is a high-resolution structure of Ssb bound to programmed ribosome\nnascent chain complexes. The authors find that the primary binding site for Ssb1 is the ribosomal protein Rpl25 (uL23).\nuL23 is at the exit tunnel and this binding site is consistent with previous crosslinking data and by convincing genetic and\nbiochemical experiments presented in this manuscript. The authors attempt to address the question of how Ssb and RAC\nfunction is coordinated by using AlphaFold predictions and proposing a complex series of domain rearrangements.\nHowever, this is highly speculative and largely leaves open the issue of the coordination between Ssb1 and RAC. Overall,\nthe experiments are cleanly executed and presented, however the manuscript would have greater impact if more concrete\nexperimental evidence were presented for the interplay between Ssb and RAC.\nMajor points\n1. Figure 1 Can the authors comment on what contributes to the differential positioning of SBD-β in the S1 and S2\nstructures? Does it make specific contacts with the ribosome or are these different conformations intrinsic to Ssb itself?\n2. In Figure 3f, the salient question is whether or not the residual ribosome binding activity of Ssb-DD1-DD2 is due to Ssz1.\nThe synthetic growth defect is suggestive but indirect. The authors should directly test if Ssz1 is responsible for the residual\nSsb binding by measuring binding of Ssb1-3*-DD1-DD2 to ribosomes or the binding of Ssb-DD1-DD2 to ribosomes in the\nabsence of Ssz1. To support the genetic interaction, the authors should also show that this protein expresses well, so that\nthe binding and growth defects cannot be attributed to poor expression of the mutant protein.\n3. In Figure 5, the authors explain that Ssb and Rac initially bind simultaneously, followed by a hand off to Ssb. They also\nargue that Ssz1 provides a binding site for Ssb1, see figure 3f. However, the data in figure 4d argue that the two complexes\nare not present on the ribosome at the same time. These results seems inconsistent. The authors generated a nice set of\ntruncated PGK1 vectors that allows the production of RNCs of different lengths. Can they show that RAC does bind on short\nnascent chains and that Ssb predominates as chains grow in length?\nMinor points\n4. Panel D of Figure 2 is conceptually unrelated to the rest of the figure and should be moved to supplementary data.\nFurthermore, the idea being tested here is that mutations in Rpl25 affect SRP binding. However, the readout is quite indirect\n- whether or not expression of a substrate for SRP is affected. Directly examining SRP binding to Rpl25-KKK ribosomes\nwould be more direct.\n5. Figure 3 introduces the DD1-DD2 mutant. Including a cartoon of this region of Ssb and the substitutions in the figure\nwould help the reader understand exactly what the mutation is without having to refer to the supplemental materials.\n6. Figure 4a Because of the scale of the x-axis, the curves are not well separated. Including an inset for ribosome\nconcentration range <1uM would be helpful.\n7. L44 Please explain the composition of RAC here rather than referring the reader to the supplemental materials.\n8. Please explain how ADP was assigned in the cryo-EM structures if the nucleotide binding domain was not resolved.\n9. In figure 5, the cartoons are too small to convey the conformational changes being proposed."}, "author_response": "REPLY TO REVIEWER COMMENTS\nWe are pleased that all three reviewers were positive regarding our manuscript. Moreover,\nwe wish to thank each reviewer for their insightful and helpful suggestions. We feel that\nby implementing the suggested changes, the manuscript has been significantly improved.\nOur replies are in blue. When appropriate, we have copied and pasted text passages (low\nres) from the revised manuscript or Supplementary Data for the Reviewers' convenience.\nReviewer #1 (Remarks to the Author):\nThe manuscript by Zhang, Grundmann et al. presents cryo-EM structures of the yeast\nHsp70 homolog Ssb bound to the ribosome. This fills a distinct knowledge gap in the field\nregarding how this protein folding chaperone can cotranslationally engage with substrates.\nThe single particle cryo-EM work is well done, and the interactions visualized in the\nstructures are nicely validated with biochemical assays and careful mutant analyses. This\nwork also convincingly demonstrates an impact of nascent protein engagement on the\nsalt-resistant binding stability of Ssb to ribosomes. Overall, these results reflect careful,\nhigh quality experimentation. However, I found the final mechanistic model to be confusing\nand unnecessarily complicated. I believe the manuscript would be improved if the authors\ndid not attempt to seemingly incorporate every single observation related to RAC and Ssb\ninto a comprehensive model and instead focused on emphasizing functional concepts that\nare clearly demonstrated versus those that may be consistent with alternative\ninterpretations.\nSpecific comments:\n1. Figure 5 presents a mechanistic model with incredibly detailed but incompletely\nsupported steps, in an apparent attempt to explain multiple conformational states of RAC\nbound to the ribosome (4 states), and separately, of Ssb bound to the ribosome (2 states).\nAlthough I appreciate the effort put into reconciling all these states, it does not seem\nnecessary, and many of the steps appear to be conjecture. In my opinion, these rather\nconfusing and detailed analyses detracts from the high-quality work in this study. I would\nfavor removing most of these descriptions and significantly simplifying the Discussion for\nseveral reasons:\na. The RAC conformational states were reported in previous studies, and there is not\nenough space to clearly explain what the four different states are. As a result, the\nDiscussion covering these states is incredibly dense with overly specialized nomenclature.\nWe agree with the above comments of Reviewer #1. In order to simplify the model and\nfocus on the central observations of this work we made the following changes:\n- We removed all hybrid RAC models (RAC S1, RAC S2, RAC S3, and RAC S4) from the\nmodel depicted in Fig. 5.\n- We now present a simplified model that involves (i) the three Ssb states that we\ncharacterize in this work: Ssb-ADP S1, Ssb-ADP S2 (cryo-EM work of this study), and\nSsb-ATP (model based on the known structure of Ssb-ATP 1 and biochemical data\npresented in this manuscript (Fig. 3e,f and Fig. 4a), (ii) the atomic model of RAC C2 (PDB\n7X3K, 2), and (iii) an AlphaFold 3 model generated with the input Ssb, Zuo1, Ssz1, which\nwe validated carefully (Supplementary Note 2 and Supplementary Fig. 10)\n1\n- We have significantly simplified the model presented in Fig. 5 as well as the Discussion\nby reducing the intermediates and by refraining from integrating all published data as\nsuggested by Reviewer #1. This allowed us to focus more clearly on the essential results\nof this work. We feel that these changes improved the value of the manuscript.\nb. It is not clear to me why all states of RAC and Ssb observed by cryo-EM must be\nconsidered functionally relevant or part of the same mechanistic pathway. There are\nnumerous examples of parallel factor-ribosome engagement pathways. Cryo-EM also\nmay capture states that are artificially stabilized during lysis and isolation procedures. It\ndoes not seem like there is sufficient evidence to suggest that all of the observed states\nmust be put into the same pathway, that they are as precisely coordinated as indicated in\nFigure 5, or that Ssb loading may not occur slightly differently for different ribosome-\nnascent protein complexes.\nWe agree with Reviewer #1 and no longer consider all cryo-EM structures (see our reply\nto comment a) above). We have revised the model depicted in Fig. 5 and adjusted the\nDiscussion accordingly. We also clarify in the Discussion that alternative pathways of the\nRAC-Ssb system may exist.\n2. The nascent protein density outside of the ribosomal exit tunnel is not clear in the\nprovided maps. Is it only visible in unsharpened maps? The maps provided also appear\nto have gone through a sharpening procedure, but sharpening B-factors are not provided\nin the statistics table.\nThe nascent chain density outside of the tunnel as shown in Fig. 1d upon submission was\nchanged according to the suggestions of Reviewer #2. Fig. 1d now shows cross sections\nthrough lowpass filtered (sdev=2.5), non-post-processed cyro-EM maps of Ssb-ADP S1\nand S2. The previous Fig. 1d is now shown as Supplementary Figure 1f.\nThe legend of Fig. 1d (*) and the Results section (**) now contain a more detailed\ndescription of the visualization of nascent chain density. Both the Ssb-ADP S1 and S2\nnon-post-processed maps, as well as the corresponding post-processed maps, are\navailable now as EMD-53860 and EMD-53861. We apologize for not mentioning the\ndeposition of the maps in our original submission. In addition, the unsharpened maps have\nbeen uploaded to the figshare repository.\n* Figure 1d\n2\n** Results\nSupplementary Fig. 1f (previous Fig. 1d)\nWith regard to the sharpening B-factor, we would like to clarify that the post-processed\nmaps were not sharpened in the classical sense. Instead, we used the tool deepEMhancer\n3, an automated deep-learning–based post-processing algorithm that does not rely on B-\nfactor estimation. Consequently, no B-factor was applied during post-processing and,\ntherefore, is not provided in the statistics table. Details can be found in Methods (***).\nSee also our reply to Reviewer #2 and Fig. 1d and Supplementary Fig. 1f.\n*** Methods:\n3. How was ADP assigned to the ribosome-bound Ssb in the structure? Was this by\ninference or by structural modeling? This reasoning could be clearer.\nWe apologize for having omitted this essential information and thank the Reviewers for\ndrawing our attention to this important point. As it was raised by all three Reviewers, our\nresponse is addressed to Reviewers #1-3.\nReviewer 1: 3. How was ADP assigned to the ribosome-bound Ssb in the structure? Was this by inference\nor by structural modeling? This reasoning could be clearer.\nReviewer 2: 3) The text indicates that Ssb is in the ADP form, but it is unclear if ADP or ATP is in the buffer\nor co-purifies with the protein. I guess the ADP form is inferred from the domain arrangement of Ssb, but this\ncould be clarified in the text.\nReviewer 3: 8. Please explain how ADP was assigned in the cryo-EM structures if the nucleotide binding\ndomain was not resolved.\nThe nucleotide binding domain (NBD) of Ssb was not resolved in the Ssb-ADP S1/S2\nstructures; therefore, the nucleotide bound to the Ssb-NBD could not be assessed directly.\nInstead, we inferred the nucleotide state from the conformation of the substrate-binding\ndomain (SBD) in the cryo-EM maps (Fig. 1b). In both Ssb-ADP structures, the\nconformation of the Ssb-SBD closely resembled that of the ADP-bound DnaK-SBD (PDB\n2KHO 4). Specifically, Ssb-SBDα (the “lid domain”) was observed in a closed position over\nthe Ssb-SBDβ substrate-binding cleft. By contrast, the SBD of ATP-bound Ssb (and\nincompatible with the conformation of ribosome-bound Ssb observed in our cryo-EM\nmaps.\nWe have improved the Introduction by providing a more detailed description of the distinct\nconformations of Hsp70 proteins and their functional significance. In the Results section,\nwe now explicitly state that the conformation of the Ssb-SBD is consistent exclusively with\nthe ADP-bound state.\nIntroduction:\nResults:\nIn the manuscript \"The cotranslational cycle of the ribosome-bound Hsp70 homolog Ssb\",\nYing Zhang and coworkers present a structure of the Hsp70 chaperone, Ssb, bound to the\nyeast ribosome. They observe good density for the SBDa domain, which allows them to\nlocalise a contact with Rpl25. This interaction interface is then tested with a series of\nexperiments involving mutagenesis and binding assays. Finally, the authors combine this\nnew structure with the previous structure of RAC to provide a theoretical model for the\ncotranslational protein biogenesis process. The structure of ribosome-bound Ssb is a\nsignificant result as it is one of the best (higher resolution) structures reported to date, to\nmy knowledge, for this important system.\nFrom the cryoEM perspective, the presented structures support the main conclusion\nregarding the interaction interface. Specifically, the cryoEM map in this region is of good\nquality, allowing the authors to place SDBDa confidently and to describe the potential\ninteraction interface between the SBDa domain and Rpl25. Namely, a complementary\nelectrostatic patch is identified on Ssb and Rpl25. The mutagenesis and binding\nexperiments that validate this potential electrostatic interaction appear to be well-\nperformed and logical.\nSpecific Comments:\n1) In the main text, the nature of the studied complex is not well explained and has to be\nunderstood from the Methods section. For example, it could be clearer that the complex\nis prepared in vitro by mixing purified protein with RNCs made from an in vitro translation\nreaction (as opposed to isolating an in vivo assembled complex). The methods also\nmention that a protein, His6-Snl1-∆TM, was also added, but it is not well explained what\nits role is, and if it is observed in the structure.\nDescription of sample preparation:\nWe thank Referee #2 for pointing that out that we need to detail the nature of Ssb-\nribosome complexes employed for cryo-EM in the Results. Please note that Ssb was not\nadded to purified RNCs, but was present during the translation reaction.\nResults:\nWe now also detail the components of the translation reactions in Methods. To drive\ntranslation, the translation buffer contained ATP, GTP, and creatine phosphate/creatine\nphosphate kinase. During subsequent FLAG-affinity purification of RNCs nucleotide was\nomitted from the buffers.\nRole of His -Snl1-∆TM\nSnl1-ΔTM during in vitro translation. The rationale was that the BAG-domain protein Snl1-\nΔTM, lacking its N-terminal transmembrane domain, was suggested to facilitate the\nrecruitment of Ssb to translating ribosomes: \"We postulate that Snl1 represents an additional method\nby which fungi recruit Hsp70 to translating ribosomes to support protein biogenesis.\" 6.\nFor this reason, we carried out in vitro translations with or without Snl1-ΔTM, isolated\nRNCs via the FLAG-tagged nascent chain, prepared cryo-EM grids, and collected single-\nparticle analysis datasets for both samples. After applying comparable particle-cleaning\nprocedures (Fig. R1), we observed that the reconstruction derived from the sample\ncontaining Snl1-ΔTM exhibited more defined density near the polypeptide exit tunnel\ncompared to the reconstruction without Snl1-ΔTM (Fig. R1). Importantly, however, we\nwere unable to detect any density corresponding to Snl1-ΔTM in the cryo-EM density\nmaps. These data (Fig. R1) suggest a positive role for Snl1-ΔTM in facilitating Ssb\nrecruitment to RNCs, possibly by enhancing either the recruitment process itself or\npromoting uniform positioning of Ssb at the tunnel exit. Nevertheless, because this\ninference is based on a single dataset and replicate, the available evidence is insufficient\nto draw a definitive conclusion. Differences in sample preparation and/or grid handling, or\ndiscrepancies during data collection offer alternative - and more likely - explanations for\nthe variation observed in both reconstructions. In the interest of scientific rigor, we\ntherefore decided to avoid overemphasizing this preliminary observation in the\nmanuscript, while taking advantage of the better quality of the Snl1-ΔTM dataset for further\nanalysis. However, we now provide more detailed information on Snl1-ΔTM in the Methods\nSection.\nmore defined density for Ssb1. The processing scheme outlines the steps used to obtain the initial 80S\nribosome consensus reconstruction, which served as the basis for downstream processing of RNCs purified\nfrom in vitro translation reactions supplemented with (a) Ssb and Snl1-ΔTM, and (b) Ssb alone. Shown are\ngold standard Fourier shell correlation plots (GSFSC plots) used for global resolution estimation, along with\nviewing angle distribution plots. Front and top views of the unsharpened (non-post-processed) maps are\npresented. Insets highlight the Ssb binding site, with Gaussian-filtered maps (sdev = 2.5) shown transparently,\nthe unsharpened maps in gray, and the Ssb1-ADP S1 model in salmon. Thresholding values used for the\nfiltered maps in ChimeraX (v.1.9) are indicated at the bottom.\nMethods:\n7\n2) It would be useful to make it clearer to the reader exactly which parts of Ssb's structure\nwere solved in relation to the ribosome. This could be facilitated by including the domain\nstructure drawing of Ssb (Extended Data Figure 1B) as part of Figure 1 and also indicating\nin the text which residues are truly solved at high resolution, which are primarily docked\nfrom a model and which are disordered. In line with this, a close-up view of the SSB density\n(i.e., using a volume zone in ChimeraX or a segmented map), coloured according to local\nresolution, with the model inside, would help convey the confidence in the SBDa model,\nwhich forms the binding interface. Similarly, the lower local resolution in SBDb could\nindicate inherent flexibility in SBDb, which could be important for function.\n- We now show the Ssb domain structure of Ssb in Fig. 1a.\n- We now show the Ssb density colored according to local resolution, with the molecular\nmodel inside, in the new Supplementary Figures 4d and 4h. These Figures reveal that\nhelix Ssb-αD, which forms the interface with Rpl25, exhibits the highest resolution within\nSsb-SBD.\n- we now state more clearly which parts of the Ssb-SBD were solved at high resolution,\nwhich were primarily docked from a model, and which are disordered.\n3) The text indicates that Ssb is in the ADP form, but it is unclear if ADP or ATP is in the\nbuffer or co-purifies with the protein. I guess the ADP form is inferred from the domain\narrangement of Ssb, but this could be clarified in the text.\nWe apologize for having omitted this essential information and thank the Reviewers for\ndrawing our attention to this important point. As it was raised by all three Reviewers, our\nresponse is addressed to Reviewers #1-3.\nor by structural modeling? This reasoning could be clearer.\nReviewer 2: 3) The text indicates that Ssb is in the ADP form, but it is unclear if ADP or ATP is in the buffer\nor co-purifies with the protein. I guess the ADP form is inferred from the domain arrangement of Ssb, but this\ncould be clarified in the text.\nReviewer 3: 8. Please explain how ADP was assigned in the cryo-EM structures if the nucleotide binding\ndomain was not resolved.\nThe nucleotide binding domain (NBD) of Ssb was not resolved in the Ssb-ADP S1/S2\nstructures; therefore, the nucleotide bound to the Ssb-NBD could not be assessed directly.\nInstead, we inferred the nucleotide state from the conformation of the substrate-binding\ndomain (SBD) in the cryo-EM maps (Fig. 1b). In both Ssb-ADP structures, the\nconformation of the Ssb-SBD closely resembled that of the ADP-bound DnaK-SBD (PDB\n2KHO 4). Specifically, Ssb-SBDα (the “lid domain”) was observed in a closed position over\nthe Ssb-SBDβ substrate-binding cleft. By contrast, the SBD of ATP-bound Ssb (and\nincompatible with the conformation of ribosome-bound Ssb observed in our cryo-EM\nmaps.\nWe have improved the Introduction by providing a more detailed description of the distinct\nconformations of Hsp70 proteins and their functional significance. In the Results section,\nwe now explicitly state that the conformation of the Ssb-SBD is consistent exclusively with\nthe ADP-bound state.\nIntroduction:\n4) Neither of the figures shows density for all 17 aa of the NC. A more complete figure\nwould help the reader understand if one can assign side chains for all 17 (line 64) or only\nbulky residues (i.e. Tyr).\nWe thank Reviewer #2 for this helpful suggestion. To reveal the side chain density of\nnascent FLAG-Pgk1-70 residues, we have now included an overlay of the Ssb-ADP S2\nFLAG-Pgk1-70 density with the corresponding atomic model in Supplementary Fig. 1e.\nSince the 3´-end of the truncated mRNA encoding FLAG-Pgk1-70 (Supplementary Fig.\n1c) is well resolved in the atomic model (Supplementary Fig. 1e) and we were able to\nassign most of the 17 amino acid side chains, the backbone of the nascent chain was\ntraced confidently. However, as Reviewer #2 correctly pointed out, not all side chains\ncould be assigned based on nascent chain density. Accordingly, we have revised the text\nto reflect this limitation.\n5) In Figure 2A, the nature of the map shown is unclear. Is it unsharpened, sharpened or\nlow-pass filtered? The sharpened map provided appears to show more details than this\nfigure. In the main text, it could be made more explicit that Figure 2A refers to State S1\nand the extended data Figure 5A to State S2, as the figures are very similar on first glance.\nThe interaction could be described better as some of the mentioned residues are\npositioned to interact directly, while others are more\nindirect.\n1. The overview maps in Figure 2a and Extended Data Figure 5a (now Supplementary\nFig. 6a) were initially created using the molmap command in ChimeraX. We have now\nreplaced these with post-processed cryo-EM maps. Additionally, the zoom into the helix\nSsb-αD/Rpl25 interface is shown from both front and back views, with the distances\nbetween residues involved in ionic interactions indicated.\n10\n2. We have improved the description of the Ssb-SBD/Rpl25 interfaces in the S1 and S2\nstates and clarified their implications more clearly in the Results section.\nWe feel that Fig. 2a, Supplementary Fig. 6a (previous Supplementary 5a), and the\ncorresponding Results section are much improved by these changes, and we thank the\nReviewer for his/her helpful comments.\n6) Line 653: It could be useful to explain how the mask was generated.\nWe have added the following text to the Methods section and provide a more detailed\nexplanation of the subsequent masking steps.\n7) Line 644: More details could be provided for the training, for example, the\nencoder/decoder dimensions and number of layers, number of epochs, and whether\nfiltering was done. In the cryodrgon step, a lot of particle projections are eliminated. Are\nthese the rotated ribosomes? Does the rotation state have a substantial effect on Ssb\nbinding, and if so, why would this be? Would it be possible to combine rotated and non-\nrotated particles by using a refinement focused on the large subunit (or maybe using\nparticle subtraction, multibody refinement)? A larger dataset could improve the density.\nHas this been tried to no effect?\nMissing parameters were added to the Methods section.\nFig. 3 to illustrate the structural variability of RNCs as revealed by cryoDRGN. Shown are\nthe clustering results for Collection 1 (Supplementary Figure 2) representative for the two\nother collections. The analysis identified several distinct clusters. The atomic models\nbased on Cluster 1 particles (Ssb-ADP S1 and S2, Fig. 1b) showed that the ribosome had\ntranslated up to the most 3′-proximal AUG codon of the truncated FLAG-Pgk1-70 mRNA\n(Fig. 1c and Supplementary Fig. 1c,e). The projections removed by clustering of the\ncryoDRGN encoded latent space are indeed rotated as well as non-rotated ribosomes\nalong the translational cycle with varying occupancy of the EPA sites by tRNAs. Also,\ndifferences in the peripheral densities corresponding to the leading or trailing ribosomes\nin a polysome were identified. These analyses demonstrated that ribosomal state - defined\nby rotation, tRNA occupancy, and polysome context - substantially affects the definition of\nSsb at the polypeptide exit tunnel. In particular, non-rotated RNCs carrying a P-site tRNA\nand accompanied by a trailing ribosome exhibited markedly increased Ssb density\ncompared to other ribosomal configurations (see Supplementary Fig. 3). In summary, in\nour experimental setup, Cluster 1 ribosomes, which are stalled, non-rotated ribosomes 7\nassociated with fully translated nascent FLAG-Pgk1-70 were the most likely candidates to\nbe associated with Ssb-ADP (see above).\nWe now better define in the cryoDRGN step in Methods and Supplementary Fig. 3:\nMethods:\nThe increased Ssb density on the stalled RNCs also explains why 3D classification of all\nparticles from the initial consensus refinement using e.g. an ellipsoid mask enclosing the\nSsb density, was without success in our hands. We hypothesize that the smaller fraction\nof Ssb bound particles in the consensus refinement precluded sufficient signal to noise\nratio for successful maximum likelihood-based 3D classification in RELION. Using\ncryoDRGN to indirectly cluster for particles with increased Ssb binding, allowed\ndownstream 3D classification in RELION.\nIndeed, increasing the dataset size can enhance the quality of cryo-EM maps. To address\nthis, we collected over 54,000 micrographs, which represents substantially more\ncompared to the datasets used in most studies. We would like to note that improvements\nin cryo-EM map quality typically follow a logarithmic trend (as shown in res-log plots),\nindicating that achieving substantial further improvements would require an order of\nmagnitude more micrographs, which is not practically feasible.\n12\n8) Figure 1D The rendering of the density in the figure does not really give it a 3D feel to\njudge if density for the NC is present or not. Moreover, the low local resolution of SBDb\ndomain, in general, could make it challenging to say the NC is there confidently (i.e. line\n63-64). That said, there is an extension (colored in red in Figure 1D) that leads out of the\nSBDb domain towards the tunnel exit. Perhaps the text \"clear nascent chain density in\nboth states\" could be rephrased as follows: \"There is an extension (red density, Figure\n1D) leading away from the SBDb density towards the NC tunnel exit, which we tentatively\nattribute to the NC.\"\nWe changed Fig. 1d according to the suggestions of Reviewer #2. Fig. 1d now shows a\ncross sections through lowpass filtered (sdev=2.5), non-post-processed cyro-EM maps of\nSsb-ADP S1 and S2. The previous Fig. 1d is now presented as Supplementary Fig. 1f.\nWe followed the suggestion of Reviewer #2 and changed the text in the Results\naccordingly:\n9) In the local resolution panels, does red indicate 6.9 Å or does it indicate > 6.9 Å? I\nmean, for example, when colouring the map, is the range set to a max of 6.9, so any voxels\nwith a value of 6.9 or above are rendered in red? What package was used for the local\nresolution calculation?\nWe thank Reviewer #2 for pointing out this mislabeling. Indeed, \"red\" indicates ≥6.9Å (3x\nNyquist limit) and we corrected Supplementary Fig. 3 (now Supplementary Fig. 4)\naccordingly. CryoSPARC was used for local resolution estimation. We included this\ninformation in the Methods section.\nMinor Comments:\n1) The company Thermo Fisher Scientific is referred to as both \"ThermoFischer Scientific\"\nand \"Thermo Scientific\" in the methods. It should be consistent and spelt correctly\nThermo Fisher Scientific is now spelled correctly throughout the text.\n2) Line 632, the provided magnification is not consistent with Table 2.\nWe thank the reviewer for pointing out this inconsistency. Magnification was 130,000x. We\ncorrected the magnification in the Methods section.\n3) Table 1 indicates the FSC threshold is 0.5, but in the Extended Data Figure 2B and E,\nit appears that 0.143 is used\nvalidation algorithm. In this procedure, theoretical maps are generated from the structural\nmodel and compared to the experimental map at an FSC threshold of 0.5 by default 8.\nHowever, the global resolution estimate, obtained by gold-standard Fourier shell\ncorrelation of the two experimental half-maps, was determined at the conventional\nthreshold of 0.143, as indicated in Supplementary Table 1 and Supplementary Fig. 4b,f.\n4) Line 807: In EMPIAR-XYZ, XYZ can be updated.\nWe updated the EMPIAR code to EMPIAR-12933 in the Data Availability section.\n5) Extended Data Fig. 2. The number of particles in the lower part of the State S1 side\nshould be double-checked. The figure indicates that 1.28x10^5 particles go into the 3D\nclassification, then 53% are selected. In the next step after the selection, the particles have\nincreased to 1.31x10^5 and again increase after the next classification to 6.14x10^5.\nWe thank the reviewer for pointing out this inconsistency. We now indicate the correct\nnumber of particles that went into the initial classification (2.43*10ˆ5) resulting the\n1.31*10ˆ5 and 6.14ˆ4 particles in the subsequent steps (Supplementary Fig. 2).\n6) Extended Data Figure 5 in some binding assays, the fractions are designated T, C, and\nR and in other T, S, and R (i.e. Extended Data Figure 7). The reason for the difference is\nunclear.\nReviewer #2 is correct; we made this distinction in the previous version of the manuscript.\nOur intention was to differentiate T (total), C (cytosol), and R (ribosomal pellet) in\nexperiments analyzing ribosome-binding of Ssb in total cell extracts from T (total), S\n(supernatant), and R (ribosomal pellet) in experiments analyzing ribosome-binding of Ssb\nusing purified components (i.e., in the absence of cytosolic proteins). However, we\nrecognize that this terminology may be confusing.\nTo clarify, we now consistently use T (total), S (supernatant), and R (ribosomal pellet)\nthroughout. Whether S refers to cytosolic proteins not associated with ribosomes or to\npurified proteins not associated with ribosomes is clearly indicated in the Figure Legends,\nand the graphs specify the y-axes as either “ribosome-bound Ssb in cell extract [%]” or\n“purified Ssb bound to purified ribosomes [%]\"\n7) Extended Data Figure 5D. Is it possible the western blots have been shifted up, as here\nSsb and rpl4 run at 116 and 66 kDa, while in Extended Data Figure 5G they run at 66 and\n35 kDa\nWe now included the correct markers for the immunoblots shown in Supplementary Fig.\n6d (previous Extended Data Fig. 5D).\n8) Extended Data Figure 4 right-hand panels. It would be useful to have the L25 label.\nWe labeled L25 (now Supplementary Fig. 5), thanks for pointing that out.\nCo-translational folding of proteins by chaperones is a critical process in protein\nhomeostasis. Hsp70s are an abundant class of chaperones that interact with and facilitate\nfolding of nascent chains. In yeast, the Hsp70 Ssb has a cochaperone, RAC, comprising\nZuo1 and SSz1. How Ssb and RAC function together on the ribosome is not well\nunderstood. In this manuscript, the authors use single particle cryo-EM as well as\nbiochemical and genetic approaches to examine this question. The principal finding of the\nmanuscript is a high-resolution structure of Ssb bound to programmed ribosome\nnascent chain complexes. The authors find that the primary binding site for Ssb1 is the ribosomal\nprotein Rpl25 (uL23). uL23 is at the exit tunnel and this binding site is consistent with\nprevious crosslinking data and by convincing genetic and biochemical experiments\npresented in this manuscript. The authors attempt to address the question of how Ssb and\nRAC function is coordinated by using AlphaFold predictions and proposing a complex\nseries of domain rearrangements. However, this is highly speculative and largely leaves\nopen the issue of the coordination between Ssb1 and RAC. Overall, the experiments are\ncleanly executed and presented, however the manuscript would have greater impact if\nmore concrete experimental evidence were presented for the interplay between Ssb and\nRAC.\nMajor points\n1. Figure 1 Can the authors comment on what contributes to the differential positioning of\nSBD-β in the S1 and S2 structures? Does it make specific contacts with the ribosome or\nare these different conformations intrinsic to Ssb itself?\nWe thank Reviewer #3 for pointing out that we had insufficiently explained this point in the\nmanuscript. To facilitate better understanding of this important result, we have revised the\nmanuscript accordingly (please see below). Overall, the conformation of the Ssb-SBD in\nthe atomic models Ssb-ADP S1 and S2 does not differ significantly. What differs is the\npositioning of the Ssb-SBDβ relative to the ribosomal surface, with S2 being approximately\n19° closer to the surface, as illustrated in Fig. 1f. Additionally, we provide a side-by-side\ncomparison (front and back views) of Ssb-ADP S1 (Supplementary Fig. 5b) and S2\n(Supplementary Fig. 5a), detailing the differences in conformation and contacts between\nthe two states.\nChanges in the manuscript:\n1.\nWe added the information that the structures of the Ssb-SBD in the S1 and S2 state are\nsimilar, however, their positioning on the ribosome differs to the Results Section.\n15\n2.\nWe improved the Figure Legends of Supplementary Figs. 4a,b (now Supplementary Fig.\n5a,b). Which now detail ribosomal contact sites of Ssb more clearly.\n16\n3. We revised Supplementary Note 1 to more clearly highlight the differences and\nsimilarities between contacts of Ssb-ADP S1 and Ssb-ADP S2.\n2. In Figure 3f (now Fig. 3g), the salient question is whether or not the residual ribosome\nbinding activity of Ssb-DD1-DD2 is due to Ssz1. The synthetic growth defect is suggestive\nbut indirect. The authors should directly test if Ssz1 is responsible for the residual\nSsb binding by measuring binding of Ssb1-3*-DD1-DD2 to ribosomes or the binding of Ssb-\nDD1-DD2 to ribosomes in the absence of Ssz1. To support the genetic interaction, the\nauthors should also show that this protein expresses well, so that the binding and growth\ndefects cannot be attributed to poor expression of the mutant protein.\nWe thank Reviewer #3 for these excellent suggestions.\nWe now show the expression level of Ssb-3*-DD1-DD2 in the new Supplementary Fig. 7d.\nThe expression of Ssb-3*-DD1-DD2 resembles the expression level of wild type Ssb.\nWe now directly compared ribosome-binding of Ssb-DD1-DD2 and ∆ssz1 Ssb-DD1-DD2\nside by side. We also included the Ssb1-3* DD1-DD2 mutant in the analysis: in this mutant\nbinding of Ssb to both Rpl25 and the Ssz1-NBD is impaired. The results are shown in the\nnew Fig. 3h and new Supplementary Fig. 7e.\nIn summary, residual ribosome binding of Ssb-DD1-DD2 (Fig. 3b,c) is further reduced\nwhen Ssb carries mutations that affect its interaction with Ssz1 or when Ssz1 is absent.\nWe have included this important finding in the Results section.\n3a. In Figure 5, the authors explain that Ssb and Rac initially bind simultaneously, followed\nby a hand off to Ssb. They also argue that Ssz1 provides a binding site for Ssb1, see figure\n3f (now 3g). However, the data in figure 4d argue that the two complexes are not present\non the ribosome at the same time. These results seems inconsistent.\nThese observations, which may initially appear contradictory, arise from markedly distinct\nconformations that Ssb adopts when bound to ATP (PDB 5TKY 5) versus when bound to\nADP (PDB 2KHO 4, and Fig. 1b of this work). The transition between these conformational\nstates, triggered by ATP hydrolysis, occurs rapidly. We have now included this essential\nbackground information more clearly in the Introduction (see below).\nThe different conformations of Ssb are depicted side-by-side in Supplementary Figs. 9a\n(ribosome-bound Ssb-ATP), 9c (ribosome-bound Ssb-ADP S1), and 9e (ribosome-bound\nSsb-ADP S2). RAC can be accommodated on ribosome-Ssb-ATP complexes\n(Supplementary Fig. 9b), and possibly also on Ssb-ADP S1 complexes; however, Ssb-\nADP S1 and RAC are already in close proximity (Supplementary Fig. 9d). RAC, however,\ncannot be accommodated on Ssb-ADP S2 complexes due to steric clashes between the\nSsz1-NBD and Ssb-SBDβ (Supplementary Fig. 9f).\nOf note, our fluorescence anisotropy measurements revealed nearly identical K values\nd\nfor Ssb binding to ribosomes and ribosome-RAC complexes when Ssb is in the open\nconformation resembling the ATP-bound state (Supplementary Fig. 8f). Moreover,\nphenotypic analysis of the Ssb-DD1-DD2 mutant, the Ssb-3*-DD1-DD2 mutant, and the\nSsb-DD1-DD2 mutant in the ∆ssz1 background (Fig. 3g), as well as analysis of ribosome\nbinding by these mutants (new Fig. 3h), are consistent with conformational states of the\nRAC-Ssb system in which RAC and Ssb can be accommodated on the same ribosome.\nHowever, after ATP hydrolysis, when Ssb is bound to ADP, Ssb and RAC can no longer\nbind concurrently to the same ribosome. This is demonstrated by the experiment shown\n18\nin Fig. 4d and is consistent with the structural models provided in Supplementary Fig. 9.\nBased on the combined data, we propose the model depicted in Fig. 5 in that RAC release\noccurs upon ATP hydrolysis by Ssb, because, in its new position, Ssb-SBDβ interferes\nwith RAC binding (Supplementary Fig. 9f). According to this model, RAC release occurs\nexactly when RAC has fulfilled its dual function: (i) positioning Ssb-SBDβ close to the\nemerging nascent chain and (ii) catalyzing ATP hydrolysis, which leads to stable binding\nof Ssb to the nascent chain - and RAC’s own release.\n3b. The authors generated a nice set of truncated PGK1 vectors that allows the production\nof RNCs of different lengths. Can they show that RAC does bind on short nascent chains\nand that Ssb predominates as chains grow in length?\nWe are pleased to refer Reviewer #3 to our previous study published in Nature Comm. in\n2020 1 under the title: \"The ribosome-associated complex RAC serves in a relay that\ndirects nascent chains to Ssb\". In Fig. 1 of Zhang et al. 2020 we show that Zuo1 and Ssz1\ncontact nascent chains prior to Ssb. For your convenience we included Fig. 1c and the\nexperimental set up, which is detailed in Fig. 1b (Zhang et al. 2020). Fig. 1c and additional\nresults of Zhang et al. 2020 reveal that the emerging nascent chain replaces the Zuo1 LP-\nmotif from the peptide binding cleft of Ssz1-SBDβ and provide functional insight into the\ncoordination between Ssb and RAC on translating ribosomes.\nFigure 1c Zhang et al. 2020 1 [redacted]\n(b) Experimental set up of crosslinking experiments. Isolated RNCs carrying [35S]-labelled nascent chains are\ncrosslinked to adjacent proteins using the homobifunctional amino-reactive crosslinker BS3, spacer length 11.4\nÅ. Crosslink products between nascent chains and Ssb, Ssz1, or Zuo1 are then identified via\nimmunoprecipitation under denaturing conditions using antibodies directed against Ssb, Ssz1, or Zuo1.\n(c) Contacts of Ssb, Zuo1, and Ssz1 with short nascent chains. RNCs carrying Pgk1-40 (40 residues), -45 (45\nresidues), or -50 (50 residues) were generated in a wild type translation extract.\nWe now present these previous findings more clearly in the Introduction, as they provide\nthe basis for the model proposed in Fig. 5.\n4. Panel D of Figure 2 is conceptually unrelated to the rest of the figure and should be\nmoved to supplementary data. Furthermore, the idea being tested here is that mutations\nin Rpl25 affect SRP binding. However, the readout is quite indirect - whether or not\nexpression of a substrate for SRP is affected. Directly examining SRP binding to Rpl25-\nKKK ribosomes would be more direct.\nWe agree with Reviewer #3 that SRP binding represents a side result in the context of this\nstudy. As requested, we moved Fig. 2d to the Supplementary Figures, where it is now\npresented as Supplementary Fig. 6i. However, we consider it important to provide an\nexplanation for the strong growth defect of the Rpl25-KKK strain (Supplementary Fig. 6b).\nThis severe growth defect is consistent with a role of Rpl25 in SRP-dependent targeting\nto the ER and is supported by the observation that Dap2, a membrane protein which\nrequires SRP for targeting, is not properly delivered in the Rpl25-KKK strain\n(Supplementary Fig. 6h).\nWith regard to directly examining SRP binding to Rpl25-KKK ribosomes, we appreciate\nthe reviewer’s suggestion. Unfortunately, with the methods currently available analysis of\nSRP binding to Rpl25-KKK ribosomes was not feasible in the context of this study for the\nfollowing reasons.\nThe bulk of SRP is lost during the preparation of total extract, which is used for ribosome-\nbinding experiments (Fig. R2). Of the residual SRP in the total extract, only a minor fraction\nis associated with ribosomes 9, likely because SRP is not stably associated with translating\nribosomes during ribosome scanning 10,11. Thus, ribosome-binding experiments, as those\nperformed with Ssb and RAC in this study, are not suited to compare the binding of SRP\nto wild type and Rpl25-KKK ribosomes. A valid comparison could be performed using in\nvitro translation followed by crosslinking and immuno-purification of the crosslinking\nproducts. We have previously performed this type of analysis with wild type translation\nextract and found that SRP is efficiently recruited to RNCs attached to Dap2, but not Pgk1\n11. However, the approach requires a translation extract prepared from the Rpl25-KKK\nstrain, which we were unable to generate due to the severe slow-growth phenotype of this\nmutant strain (Supplementary Fig. 6b).\n(yeast cells lysed under denaturating conditions) was prepared as described in the manuscript. Loading was\nadjusted to equalize ribosome loading (Rpl4).\nTo acknowledge the limitations of our SRP data, we have carefully revised and moderated\nour statement in the Results section and more clearly indicated the purpose of the SRP-\nrelated experiments. Please note that we do not claim that SRP does not bind to Rpl25-\nKKK ribosomes, but rather focus on the effect that could underlie slow growth of the Rpl25-\nKKK strain.\n5. Figure 3 introduces the DD1-DD2 mutant. Including a cartoon of this region of Ssb and\nthe substitutions in the figure would help the reader understand exactly what the mutation\nis without having to refer to the supplemental materials.\nThis is an excellent suggestion. We now show a cartoon of helix SBD-αD as Fig. 3a.\n6. Figure 4a Because of the scale of the x-axis, the curves are not well separated. Including\nan inset for ribosome concentration range <1uM would be helpful.\nReviewer #3 is correct. We initially tried using an inset, but it provided little improvement.\nWe now display the x-axis on a logarithmic scale as typical in the field. The inflection point\nof the sigmoidal curves on the log-scaled axis allows an approximate visual estimation of\nthe K .\nd\n21\n7. L44 Please explain the composition of RAC here rather than referring the reader to the\nsupplemental materials.\nWe have extended the Introduction to provide a more detailed description of the structure\nof RAC.\n8. Please explain how ADP was assigned in the cryo-EM structures if the nucleotide binding\ndomain was not resolved.\nWe apologize for having omitted this essential information and thank the Reviewers for\ndrawing our attention to this important point. As it was raised by all three Reviewers, our\nresponse is addressed to Reviewers #1-3.\nReviewer 1: 3. How was ADP assigned to the ribosome-bound Ssb in the structure? Was this by inference\nor by structural modeling? This reasoning could be clearer.\nReviewer 2: 3) The text indicates that Ssb is in the ADP form, but it is unclear if ADP or ATP is in the buffer\nor co-purifies with the protein. I guess the ADP form is inferred from the domain arrangement of Ssb, but this\ncould be clarified in the text.\nReviewer 3: 8. Please explain how ADP was assigned in the cryo-EM structures if the nucleotide binding\ndomain was not resolved.\nThe nucleotide binding domain (NBD) of Ssb was not resolved in the Ssb-ADP S1/S2\nstructures; therefore, the nucleotide bound to the Ssb-NBD could not be assessed directly.\nInstead, we inferred the nucleotide state from the conformation of the substrate-binding\ndomain (SBD) in the cryo-EM maps (Fig. 1b). In both Ssb-ADP structures, the\nconformation of the Ssb-SBD closely resembled that of the ADP-bound DnaK-SBD (PDB\n2KHO 4). Specifically, Ssb-SBDα (the “lid domain”) was observed in a closed position over\nthe Ssb-SBDβ substrate-binding cleft. By contrast, the SBD of ATP-bound Ssb (and\nincompatible with the conformation of ribosome-bound Ssb observed in our cryo-EM\nmaps.\nWe have improved the Introduction by providing a more detailed description of the distinct\nconformations of Hsp70 proteins and their functional significance. In the Results section,\nwe now explicitly state that the conformation of the Ssb-SBD is consistent exclusively with\nthe ADP-bound state.\nIntroduction:\nResults:\n9. In figure 5, the cartoons are too small to convey the conformational changes being\nproposed.\nWe have enhanced the size of the cartoons in the model shown in Fig. 5 and also simplified\nthe model significantly. Detailed views of ribosome-bound RAC and Ssb in the different\nconformational states are also displayed in Supplementary Figs. 9 and 10.\nDuring the revision process, we corrected the molecular models of Ssb-ADP S1 and S2.\nThe C-terminal carboxyl group of Rpl25 had been mistakenly modeled as a hydroxyl\ngroup; this error has been corrected. The Rpl25 C-terminal carboxyl group is important for\nthe interaction between Rpl25 and Ssb (Fig. 2a and Supplementary Fig. 6a). Furthermore,\nin the initial ribosome model (PDB 6T7I), incorrect sequences were linked to Rpl6b and\nRpl24b (Fig. R3). The Rpl6b and Rpl24b sequences have been been corrected. The\nrevised models have been re-uploaded to the PDB, and Supplementary Table 1 has been\nupdated accordingly.\nFigure R3. Clustal alignment of (a) Rpl6b and (b) Rpl24b sequences in PDB 6T7I and the corresponding\nsequence from UniProt.\n⍵\n1. Zhang, Y. et al. The ribosome-associated complex RAC serves in a relay that directs\nnascent chains to Ssb. Nat. Commun. 11, 1504 (2020).\n2. Chen, Y., Tsai, B., Li, N. & Gao, N. Structural remodeling of ribosome associated Hsp40-\nHsp70 chaperones during co-translational folding. Nat. Commun. 13, 3410 (2022).\n3. Sanchez-Garcia, R. et al. DeepEMhancer: a deep learning solution for cryo-EM volume\npost-processing. Commun Biol 4, 874 (2021).\n4. Bertelsen, E. B., Chang, L., Gestwicki, J. E. & Zuiderweg, E. R. Solution conformation of\nwild-type E. coli Hsp70 (DnaK) chaperone complexed with ADP and substrate. Proc. Natl.\nAcad. Sci. U S A 106, 8471-8476 (2009).\n5. Gumiero, A. et al. Interaction of the cotranslational Hsp70 Ssb with ribosomal proteins and\nrRNA depends on its lid domain. Nat. Commun. 7, 1-12 (2016).\n6. Verghese, J. & Morano, K. A. A lysine-rich region within fungal BAG domain-containing\nproteins mediates a novel association with ribosomes. Eukaryot. Cell 11, 1003-1011\n(2012).\n7. Tesina, P. et al. Molecular mechanism of translational stalling by inhibitory codon\ncombinations and poly(A) tracts. EMBO J. 39, e103365 (2019).\n8. Afonine, P. V. et al. New tools for the analysis and validation of cryo-EM maps and atomic\nmodels. Acta Crystallogr D Struct Biol 74, 814-840 (2018).\n9. Raue, U., Oellerer, S. & Rospert, S. Association of protein biogenesis factors at the yeast\nribosomal tunnel exit is affected by the translational status and nascent polypeptide\nsequence. J. Biol. Chem. 282, 7809-7816 (2007).\n10. Flanagan, J. J. et al. Signal recognition particle binds to ribosome-bound signal sequences\nwith fluorescence-detected subnanomolar affinity that does not diminish as the nascent\nchain lengthens. J. Biol. Chem. 278, 18628-18637 (2003).\n11. Berndt, U., Oellerer, S., Zhang, Y., Johnson, A. E. & Rospert, S. A signal-anchor sequence\nstimulates signal recognition particle binding to ribosomes from inside the exit tunnel. Proc.\nNatl. Acad. Sci. U S A 106, 1398-1403 (2009).\n25"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised manuscript addresses my comments and I support publication.", "reviewer_2": "I would like to thank the authors for their thorough response and revision. The manuscript has\nimproved in both clarity and rigour. From my side, the responses to the review comments are\ndetailed and convincing, and I find that my previous concerns have been largely addressed. I\nhave no further major comments.\nA minor question/clarification would be how the rotation axis in Supplementary Fig. 3D was\ndetermined. Also in Figure 3 are the volumes shown from a backprojection (cryodrgn\nbackproject_voxel) of all particles in the class, or the volume at the centre of the class?"}, "author_response": "Reply to Reviewers\nReviewer #1 (Remarks to the Author):\nThe revised manuscript addresses my comments and I support publication.\nWe thank Reviewer #1 for his/her positive evaluation.\nReviewer #2 (Remarks to the Author):\nI would like to thank the authors for their thorough response and revision. The manuscript has\nimproved in both clarity and rigour. From my side, the responses to the review comments are\ndetailed and convincing, and I find that my previous concerns have been largely addressed. I\nhave no further major comments.\nWe thank Reviewer #2 for his/her positive response. We also feel that the manuscript is\nimproved thanks to the input of the Reviewers.\nA minor question/clarification would be how the rotation axis in Supplementary Fig. 3D was\ndetermined. Also in Figure 3 are the volumes shown from a backprojection (cryodrgn\nbackproject_voxel) of all particles in the class, or the volume at the centre of the class?\n1. Determination of the rotation axis\nThe axes connect the tip of expansion segment 6 (ES6) of the 18S rRNA to the C-terminus of\nthe 40S ribosomal protein Asc1. The black axis represents the orientation in the non-rotated\nstate, and the gray axis shows the rotation of the 40S subunit relative to this state, with the\n60S subunit held fixed.\nWe now specify how the rotated axis was determined in the Legend of Supplementary Fig. 3d.\n(d) Rotational state of the 40S ribosomal subunit in clusters 1 to 8. The axes connect the tip of\nexpansion segment 6 (ES6) of the 18S rRNA to the C-terminus of the 40S ribosomal protein\nAsc1. Non-rotated axis (black line), rotated axis (gray line), with the 60S subunit held fixed.\n2. cryoDRGN analysis\nIn the cryoDRGN analysis, we performed kmeans clustering on the particles latent space\nembeddings. For this purpose, 1000 volumes were generated via back projection of all\nparticles within a cluster. Subsequently, these 1000 reconstructed volumes were clustered\nusing the agglomerative clustering approach of the cryoDRGN landscape analysis tool. The\nvolumes shown in Supplementary Fig. 3 are centroids, representing a voxel-based average of\nall reconstructions that were grouped by agglomerative clustering.\nWe now explain the volume generation in more detail in the Methods section.\nUsing the cryoDRGN landscape’ tool, latent space encodings were first clustered via kmeans\n(k = 1000) and the resulting reconstructions, generated by back projection, were further\nclustered via agglomerative clustering (k = 20) (https://ez-lab.gitbook.io/cryodrgn/). The\nvolumes of the resulting clusters are shown as centroids, representing voxel-based averages\nof the reconstructions within each cluster (Supplementary Fig. 3a,b)."}]

Decision letter