Structure of the mammalian ribosome-Sec61 complex to 3.4 Å resolution

Abstract

Cotranslational protein translocation is a universally conserved process for secretory and membrane protein biosynthesis. Nascent polypeptides emerging from a translating ribosome are either transported across or inserted into the membrane via the ribosome-bound Sec61 channel. Here, we report structures of a mammalian ribosome-Sec61 complex in both idle and translating states, determined to 3.4 and 3.9 Å resolution. The data sets permit building of a near-complete atomic model of the mammalian ribosome, visualization of A/P and P/E hybrid-state tRNAs, and analysis of a nascent polypeptide in the exit tunnel. Unprecedented chemical detail is observed for both the ribosome-Sec61 interaction and the conformational state of Sec61 upon ribosome binding. Comparison of the maps from idle and translating complexes suggests how conformational changes to the Sec61 channel could facilitate translocation of a secreted polypeptide. The high-resolution structure of the mammalian ribosome-Sec61 complex provides a valuable reference for future functional and structural studies.

Graphical abstract

Figure 1

The Structure of a Mammalian Ribosome-Translocon Complex (A) Model of the idle 80S ribosome in complex with Sec61, shown in red. The color scheme shown here is used throughout the manuscript: 40S rRNA is displayed in orange, the 40S ribosomal proteins in brown, the 60S rRNA in cyan, and the 60S ribosomal proteins in dark blue. The region of the peptidyl transferase center (PTC) is indicated. (B) Cut view of the final unsharpened cryo-EM density map for both the idle 60S-Sec61 complex and the 40S subunit, colored by local resolution in Å (Kucukelbir et al., 2014). Also see Figure S3.

Figure 2

Representative Density for the Ribosomal Proteins and rRNA (A–D) Cryo-EM density for the 60S subunit and the body of the 40S was sufficient to allow unambiguous placement of rRNA bases (A, C, D) amino acid side chains (B, C, D), and many ions (D). Also see Figure S4.

Figure 3

An A/P Hybrid State tRNA (A) Overview of the hybrid A/P (purple) and P/E tRNAs (green) visualized in the translating ribosome-Sec61 structure. (B and C) Adoption of the hybrid A/P conformation (purple) relative to the canonical A-site tRNA (gray) requires a ∼13° rotation in the backbone of the tRNA just above the anticodon stem loop, as well as a 10° rotation in the acceptor/T-stem stack and a 9 Å displacement of the 3′ tail.

Figure 4

Interaction of Sec61 with the Ribosome (A) Overview of the region of the ribosome surrounding the Sec61 complex, including the cytosolic loops 6/7 and 8/9. Sec61α is displayed in red, γ in tan, and β in light blue. (B) Close-up of the cytosolic loops of Sec61 and the surrounding ribosomal proteins and RNA. (C and D) Representative density for the cytosolic loops of Sec61α, regions of Sec61γ, and their corresponding helices. (E) Hydrogen bonding interactions between residues H404 and R405 in loop 8/9 of Sec61α and ribosomal protein uL23 and the 28S rRNA. (F) Visualization of a salt bridge between R20 in the N-terminal helix of Sec61γ and D148 in υΛ23. (G) An arginine stack between residue R273 in loop 6/7 and R21 in eL39 is stabilized by interaction with the backbone of the 28S rRNA. See also Figure S5.

Figure 5

Conformation of Ribosome-Bound Sec61α (A) Overview of the lateral gate of the ribosome-bound Sec61α in red, compared to the isolated crystal structure of the archaeal SecY in gray (Park et al., 2014). (B) Cytosolic loop 6/7 shifts by 11 Å relative to the the archaeal SecY structure. (C) Cytosolic loop 8/9 shifts by 6 Å relative to the bacterial SecY structure shown in pink (Tsukazaki et al., 2008). The bacterial structure is used for comparison here because loop 8/9 is disordered in the archaeal structure. (D) Close-up of the lateral gate (helices 2 and 3 with helices 7 and 8), highlighting the opening of the cytosolic region between helices 8 and 2 in the ribosome-bound state. (E) Close-up of the plug region, which is unaltered in the ribosome-bound state. (F) Comparison of the lateral gate in the Sec61-ribosome structure relative to that observed in the SecY-SecA complex (light blue; Zimmer et al., 2008).

Figure 6

The Translating Ribosome-Sec61 Complex (A) Cryo-EM density within the ribosomal exit tunnel for the nascent peptide (cyan), which spans from the A/P tRNA to Sec61. The location of ribosomal protein eL39, which lines the exit tunnel, is indicated. (B) Ribosomal protein eL39 (bright blue) forms part of the exit tunnel (highlighted in cyan) and interacts with loop 6/7 of Sec61. Ribosomal protein uL23 (dark blue) contacts both eL39 and loop 8/9 of Sec61. (C) Comparison of the Sec61 channel structures bound to idle or translating ribosome, showing movements in helices 1 and 10, which may be important for allowing translocation of the nascent polypeptide. Also see Figure S6. (D) Rigid-body fitting of the idle Sec61 model (red) into the density for the translating Sec61-ribosome complex demonstrates that the plug is not visible in its canonical location. Displayed is an unsharpened map in which the disordered density for the detergent micelle has been removed using Chimera (Goddard et al., 2007).

Figure 7

A Two-Step Model for Activation of Sec61 Displayed here is a cut-away view of the model for the Sec channel from the central pore toward the lateral gate (dashed line). In the quiescent state (left), approximated by a crystal structure of the archaeal SecY complex (Van den Berg et al., 2004), the Sec channel is closed to both the lumen and lipid bilayer. The channel becomes primed for protein translocation upon ribosome binding (middle), triggering conformational changes in Sec61 that crack the cytosolic side of the lateral gate (demarcated by an asterisk). The movements of helices 2 and 3 in this region may create an initial binding site for signal peptide recognition. Engagement of the lateral gate by the signal peptide would open the channel toward the membrane and initiate translocation (not depicted; Park et al., 2014). The translocating state of the active ribosome-Sec61 complex (right) contains a nascent polypeptide (teal) and is characterized by a dynamic plug domain and an open conduit between the cytosol and lumen (teal dotted line).

Figure S1

Biochemical Characterization of the Ribosome-Sec61 Sample, Related to Experimental Procedures (A) Immunoblot using anti-puromycin (DHSB anti-PYM 24As) of untreated and puromycin treated porcine pancreas microsomes. The ponceau stained blot is shown on the left. (B) Puromycin-treated pancreas microsomes (total) were extracted with buffer containing 0.1 M HEPES (pH 7.5), or 0.1 M Na₂CO₃ (pH 11.5), and subjected to ultracentrifugation (Fujiki et al., 1982). The supernatant (S) and membrane pellet (P) were analyzed by anti-puromycin immunoblot, coomassie staining, or immunoblot against the indicated integral membrane proteins. (C) Pancreas microsomes were solubilised with digitonin, clarified of insoluble material, and the extracted proteins separated by gel filtration using Sephacryl S-300 exactly as for samples prepared for cryo-EM analysis. Aliquots of the starting sample, soluble extract, and gel filtration fractions were analyzed by SDS-PAGE and coomassie staining. The asterisks show the ribosome-containing void fractions. (D) A digitonin-soluble extract prepared as in (C) was divided in two, fractionated by Sephacryl S-300 or 10%–50% sucrose gradient (as in Shao et al., 2013), and the fractions analyzed by immunoblotting for subunits of the OST, TRAP, and Sec61 complexes (antibodies characterized in Fons et al., 2003). The peak ribosomal fraction is indicated by absorbance at 260 nm. Note that most of OST and a portion of TRAP seem to dissociate during sucrose gradient fractionation.

Figure S2

Refinement and 3D Classification Strategy, Related to Experimental Procedures Each class in the displayed flowchart shows a cross section image of the respective map, the derived model, any mask that was applied (red dashed line), and the structure (s) derived from that class (red text). The complete data set containing 80,019 particles was refined using RELION (Scheres, 2012a, 2012b), resulting in an initial reconstruction calculated to 3.4 Å resolution (Map 1). As the 40S subunit was in a variety of distinct conformations, a soft mask for the 60S subunit was used throughout the refinement procedure (Map 2). Although this only resulted in a modest nominal increase in resolution (3.35 Å), the observed density for the 60S subunit was improved compared to the complete refinement, and was used to build the 60S ribosomal proteins and RNA. In parallel, 3D classification of the entire data set identified 13% of particles containing hybrid A/P- and P/E-site tRNAs and a nascent peptide. In order to supplement this relatively small class, an additional data set was collected, resulting in a combined14,723 particles used to build the translating ribosome-Sec61 complex to 3.9 Å resolution (Map 3). The remaining 87% of particles (69,464) were used to model the idle ribosome-Sec61 complex, lacking a nascent chain and tRNAs, to 3.4 Å resolution (Map 4). Within this idle class, 36,667 particles containing eEF2, which stabilized both the stalk base proteins and the orientation of the 40S subunit relative to the 60S (Map 5). This class of particles was used to build a model for the 40S ribosomal proteins and RNA at 3.5 Å resolution. The remaining idle particles either contained eEF2 bound in different ratcheted conformations (19%) or were devoid of tRNAs or factors (22%). All reported resolutions are determined using the gold-standard Fourier Shell Correlation (FSC = 0.143) criterion (Scheres and Chen, 2012), and are shown in Figure S3.

Figure S3

Map and Model Quality, Related to Figure 1 (A) Gold-standard Fourier Shell Correlation (FSC) curve for the map used for building of the 60S subunit (60S-mask, Map 2), where the resolution is demarcated using the FSC = 0.143 criterion. (B) FSC curves of the final model versus the full map used in A (black); of a model refined in the first of two independent halves of the map (red); and of that same model versus the second independent map, which was not used for refinement (blue). The vertical dashed line indicates the highest resolution used in these model refinements. (C and D) As in (A) and (B) but for the 40S subunit built using Map 5. The final refined models for both the 40S and 60S subunits were rigid-body fit into the density for each of the other classes of particles. (E and F) FSC curves for maps of the remaining classes.

Figure S4

Examples of Revised and Newly Visible Ribosome Features, Related to Figure 2 (A) An example of a small change in registry in the N terminus of ribosomal protein eL32 (light pink; PDB ID 3J3F; Anger et al., 2013) that could be clearly visualized and remodelled (dark blue) using the improved density map. (B) An example of a mammalian specific expansion to ribosomal protein eL6 that could be modeled unambiguously using the new data. (C) Overview of binding of eEF2 in a half-ratcheted conformation, as calculated to 3.5 Å resolution using 36,667 particles. (D) Cryo-EM density for eEF2 and its interaction partners in the ribosome stalk were well-defined, allowing building of many side chains in the ribosomal proteins and GTPase. The chemical interaction between eEF2 and ribosomal proteins uL10 and uL11 could thus be visualized.

Figure S5

Density in Different Regions of the idle Sec61 Structure, Related to Figure 4 (A) Displayed here are two views of a density map filtered to 4.2 Å resolution that demonstrates the overall fit of the idle Sec61 channel. (B) Representative density for the N terminus of Sec61γ (tan), which was sufficiently ordered to allow placement of amino acid side chains. (C) Displayed is an unsharpened density map, in which the detergent micelle has been removed using Chimera (Goddard et al., 2007), visualized from the lumenal side of the channel to show placement of the backbone of the plug in the idle channel.

Figure S6

Density and Features of the Sec61 Structure Bound to the Translating Ribosome, Related to Figure 6 (A) Displayed is an unsharpened density map, in which the detergent micelle has been removed, to demonstrate the overall fit for the translating Sec61 channel. The same views as Figure S5A are shown for comparison. (B) Displayed is a sharpened map for the cytosolic loops of the translating ribosome-Sec61 structure. Though regions of the map are only visible to moderate resolution, areas of Sec61 that interact with the ribosome are well ordered and allow placement of amino acid side chains. (C) Model of the translating ribosome-bound Sec61α colored by atomic B-factor, with regions of lowest B-factor displayed in blue, and the highest in red. The same views as shown in (A) are displayed for comparison.