Most eukaryotic secretory and membrane proteins are translocated co-translationally across the endoplasmic reticulum (ER) membrane at a ribosome-translocon complex (RTC). The central component of this translocon is the Sec61 complex, a heterotrimer containing a channel-forming α subunit and peripheral β and γ subunits (Van den Berg et al., 2004; Rapoport et al., 2017). In prokaryotes, the homologous SecYEG complex mediates translocation across the plasma membrane. In all organisms, this central channel associates dynamically with a variety of partners and accessory factors (Gemmer and Förster, 2020; Shao, 2023; Hegde and Keenan, 2022b; Smalinskaitė and Hegde, 2023). The structure and function of some accessory factors, such as the oligosaccharyl transferase complex (OST), are well established (Braunger et al., 2018), whereas many others are poorly understood (Gemmer and Förster, 2020; Shao, 2023; Hegde and Keenan, 2022b; Smalinskaitė and Hegde, 2023). Although these various translocon components have long been speculated to engage in a context- and substrate-dependent manner (Johnson and van Waes, 1999; Hegde and Kang, 2008), the rules governing their coordination are largely unclear and only now beginning to emerge (Smalinskaitė et al., 2022; Sundaram et al., 2022).

Sec61α is a pseudosymmetric channel that can open axially across the membrane and is laterally gated towards the lipid bilayer (Van den Berg et al., 2004; Rapoport et al., 2017). In its closed state, the axial channel’s pore is constricted, blocked by a short helix known as the plug, and the lateral gate is closed. Transport of hydrophilic polypeptide segments through Sec61 can be initiated by a flanking hydrophobic α-helix. These hydrophobic helices act as signal sequences that bind to Sec61’s lateral gate (Voorhees and Hegde, 2016a; Li et al., 2016). This binding is thought to widen the central pore, destabilise the plug, and thread one of the signal’s hydrophilic flanking regions into the channel to initiate translocation. A set of hydrophobic residues, known as the pore ring, lines the narrowest part of the channel and forms a gasket-like seal that maintains the permeability barrier during translocation (Dalal and Duong, 2009; Park and Rapoport, 2011; Ma et al., 2019).

This model of channel opening is derived from structures of a cleavable signal peptide (SP) bound to the mammalian Sec61 complex (Voorhees and Hegde, 2016a) or bacterial SecYEG (Li et al., 2016; Ma et al., 2019). Most membrane proteins lack an N-terminal SP and instead initiate translocation using their first TMD, often termed a signal anchor (SA). Although it has long been thought an SA initiates flanking domain translocation the same way as an SP (High et al., 1993a; Görlich and Rapoport, 1993; Mothes et al., 1998; Heinrich et al., 2000), direct evidence for this idea is sparse and somewhat contradictory. Furthermore, the route an SP or SA takes to the lateral gate remains speculative. One model posits that they all access the lateral gate from the channel interior, displacing the plug en route. Alternatively, a hydrophobic helix could slide along the lipid-facing side of the lateral gate (Cymer et al., 2015), or use a member of the Oxa1 family for insertion (Anghel et al., 2017; Chitwood et al., 2018; Wu and Hegde, 2023). Structures of TMD insertion intermediates would help resolve these and other crucial mechanistic issues in membrane protein biogenesis.

Beyond the Sec61 channel, a number of accessory factors co-translationally modify the polypeptide, facilitate membrane protein biogenesis, or facilitate secretion through Sec61. The two major modification factors are OST (Braunger et al., 2018) and the signal peptidase complex (SPC) (Liaci et al., 2021), which mediate co-translational N-linked glycosylation and SP cleavage, respectively. Based on recent structural and functional studies (Smalinskaitė et al., 2022; Sundaram et al., 2022; Chitwood et al., 2018; Wu and Hegde, 2023; McGilvray et al., 2020; Chitwood and Hegde, 2020; Shurtleff et al., 2018; Gemmer et al., 2023b), and consistent with genetic co-dependency analysis across more than a thousand cancer cell lines (Meyers et al., 2017; Wainberg et al., 2021; Dempster et al., 2019), four protein complexes are now assigned to co-translational membrane protein biogenesis: EMC, PAT, GEL, and BOS. The functions of these complexes and the dynamics of their association with nascent substrates at the ribosome are not well understood.

EMC is a 9-protein complex conserved across eukaryotes and implicated in diverse aspects of membrane protein biogenesis (Hegde, 2022a). At least one main function of EMC is to facilitate insertion of TMDs close to the N- or C-terminus. This includes post-translational insertion of tail-anchored proteins (Guna et al., 2018), post-translational insertion of the final TMD of some multipass membrane proteins (Wu et al., 2024), and co-translational insertion of SAs in the N_exo topology (N-terminus facing the exoplasmic side of the membrane) (Chitwood et al., 2018; O’Keefe et al., 2021). This insertase function is thought to involve its core EMC3 subunit (Wu and Hegde, 2023; Wu et al., 2024; Pleiner et al., 2020; Miller-Vedam et al., 2020; Bai et al., 2020; Güngör et al., 2022), which is a member of the Oxa1 family of insertases (Anghel et al., 2017). Although EMC has been implicated in co-translational membrane protein insertion, it has thus far not been observed at RTCs in cryo-electron tomography studies (Gemmer et al., 2023b; Gemmer et al., 2023a).

The PAT, GEL, and BOS complexes are recruited to the ribosome-Sec61 complex at a later stage of multipass biogenesis to form the multipass translocon (MPT) (Smalinskaitė et al., 2022; Sundaram et al., 2022; McGilvray et al., 2020; Chitwood and Hegde, 2020). The MPT is thought to facilitate membrane protein biogenesis by a combination of TMD insertion, chaperoning, and shielding (Hegde and Keenan, 2022b; Smalinskaitė and Hegde, 2023). The MPT-mediated insertion reaction is thought to operate on pairs of TMDs connected by a short translocated loop. This insertion might be facilitated by the GEL complex, whose TMCO1 subunit is another member of the Oxa1 family (Anghel et al., 2017), and does not seem to rely on the lateral gate of the Sec61 complex (Smalinskaitė et al., 2022). TMDs with partially polar character are thought to be chaperoned by the PAT complex (Chitwood and Hegde, 2020) via an amphiphilic surface on its Asterix subunit (Smalinskaitė et al., 2022). The function of the BOS complex is unclear, but its contacts with Sec61 and the ribosome may facilitate MPT assembly or stability (Smalinskaitė et al., 2022; McGilvray et al., 2020). The PAT, GEL, and BOS complexes together form a horseshoe-shaped lipid-filled cavity (Smalinskaitė et al., 2022; McGilvray et al., 2020; Gemmer et al., 2023b; Gemmer et al., 2023a) speculated to be the site of multipass membrane protein folding (Hegde and Keenan, 2022b; Smalinskaitė and Hegde, 2023).

Three factors are linked to co-translational Sec61-mediated secretion: TRAM (Görlich et al., 1992; Voigt et al., 1996; Hegde et al., 1998), TRAP (Fons et al., 2003), and RAMP4 (also called SERP1, known as Ysy6 in yeast) (Görlich and Rapoport, 1993; Schröder et al., 1999; Yamaguchi et al., 1999; Sakaguchi et al., 1991). Both TRAP and RAMP4 are tightly associated near-stoichiometrically with ER-localised ribosome-Sec61 complexes from pancreas (Görlich and Rapoport, 1993), an exceptionally secretory tissue. TRAM, although not tightly associated with ribosomes, can be crosslinked to SPs and TMDs at a point when they are at Sec61’s lateral gate (Görlich et al., 1992; High et al., 1993b; Jungnickel and Rapoport, 1995). Depletion of TRAM or TRAP reduces the capability of some SPs to successfully initiate translocation through Sec61 in vitro, placing their function at an early gating step (Görlich et al., 1992; Voigt et al., 1996; Fons et al., 2003). The role of RAMP4 is largely unclear, but it can be crosslinked to nascent chains translocating through Sec61 (Schröder et al., 1999), shows secretion defects in knockout cells and mice (Hori et al., 2006), and it is induced by ER stress (Yamaguchi et al., 1999). The mechanisms by which any of these factors impacts secretion are unclear, but would be aided by structural information on how they engage the ribosome-Sec61 translocon.

In this study, we have taken advantage of the heterogeneity of most stalled protein biogenesis intermediates assembled in vitro. Whereas substrates at certain key steps might have uniform and stable interactions with the biogenesis machinery (Voorhees and Hegde, 2016a), others probably sample multiple states dynamically (Sundaram et al., 2022). For example, both OST and MPT factors dynamically and heterogeneously associate at their overlapping sites behind Sec61. TMDs sample multiple environments such as the lateral gate, intramembrane chaperones, and the surrounding lipid. Hence, a substrate stalled at a single point during elongation can form multiple RTCs. Improvements in cryo-electron microscopy (cryo-EM) imaging and particle classification now allow sample heterogeneity to be resolved into multiple discrete density maps (Scheres, 2016; von Loeffelholz et al., 2017). When combined with rapid advances in structure prediction (Jumper et al., 2021; Evans et al., 2021; Mirdita et al., 2022), these maps can be fitted with reliable models. Using this approach, we present structures of a TMD insertion intermediate at Sec61’s lateral gate, RAMP4 and TRAP within RTCs, and a new configuration of ribosomal protein uL22 that forms contacts with Sec61 and the nascent substrate.

We performed a thorough single-particle cryo-EM analysis of a previous dataset of RTCs engaged in biogenesis of the multipass membrane protein rhodopsin (Rho) (Smalinskaitė et al., 2022). The construct is termed Rho^ext because it contains the first two TMDs of Rho and is extended at its N-terminus by fusion to an SP and an epitope tag (Figure 1A). The Rho^ext intermediate in this sample has elongated to the point where the SP has been removed after directing translocation of the N-terminal domain across the membrane, RhoTM1 has inserted into the membrane, and RhoTM2 has emerged from the ribosome (Smalinskaitė et al., 2022). At this critical chain length, the nascent chain is poised at a point where it can potentially form several different RTCs (Figure 1B–E; Figure 1—figure supplement 1), only one of which was analysed previously (Smalinskaitė et al., 2022).

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Crosslinking and co-association experiments (Smalinskaitė et al., 2022; Sundaram et al., 2022; Chitwood and Hegde, 2020) show that the nascent chain is just long enough for RhoTM1 to begin recruiting the PAT complex and initiate assembly of the MPT. Analysis of this subset of RTCs previously showed how the PAT complex latches Sec61 shut and redirects RhoTM2 towards the just-assembling MPT (Figure 1E; Smalinskaitė et al., 2022). Because PAT complex recruitment and MPT assembly are just beginning, the dataset also contains multiple PAT-free complexes. We now analyse these particles via extensive classification (Figure 1—figure supplement 1) and find that without PAT, Sec61 is either closed, opened by RhoTM2, or opened by an unexpected factor, RAMP4 (Figure 1B–D). The closed structure is similar to the previously reported PAT-bound structure, so we focus on RhoTM2- and RAMP4-bound structures. We then compare various available structures, together with structure predictions and modelling, to reveal new insights into the structural dynamic of RTCs.

The structure of Sec61 inserting a membrane protein

In the absence of the PAT complex, the Sec61 complex is free to open. In the intermediate being analysed, RhoTM2, whose eventual topology in the final protein would be N_cyt (i.e. the N-terminal flanking domain facing the cytosol), has just fully emerged from the ribosome with a 33 amino acid downstream tether to the P site tRNA. This is long enough for the first half of RhoTM2 to begin engaging the Sec61 complex, and a subset of RTCs displayed this event (Figure 2A and B). We find that the N-terminal half of RhoTM2 binds to Sec61 very similarly to the previously characterised hydrophobic helix (h-region) of an SP (Figure 2C; Voorhees and Hegde, 2016a; Li et al., 2016). Thus, both kinds of N_cyt substrate, SPs and TMDs, can bind Sec61 in the same way, despite TMDs having much longer hydrophobic helices than SPs do. The extra length of this TMD is passing through the channel pore, as will be discussed below.

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Bound in this position, the N-terminal end of the hydrophobic region of the SP or TMD is only 11 Å (∼3 aa) from the tip of 28S rRNA helix 59 (H59) and the adjacent polyacidic tail of eL22 (Figure 2B). The backbone phosphates and the exposed aromatic rings of U2707-8 in H59, and the acidic residues of eL22, are potential binding sites for basic side chains. Importantly, basic resides are sharply enriched ~3–5 aa from the hydrophobic helix (Baker et al., 2017). Thus, H59 and eL22 are well positioned to engage cationic residues flanking a signal and retain them in the cytosol, a phenomenon called the positive-inside rule (Nilsson et al., 2005). Notably, the next nearest rRNA, H47, is too far to serve this role (32 Å, ~9 aa). Earlier lower resolution maps of a bacterial RTC similarly noted the proximity of H59 to the membrane and SecY’s lateral gate (Frauenfeld et al., 2011). Given that H59 is also near the N-terminus of an SP bound to bacterial and mammalian signal recognition particle (SRP) (Jomaa et al., 2016; Kobayashi et al., 2018), H59 may contribute to the positive-inside rule throughout both targeting by SRP and insertion by Sec61.

To accommodate the TMD, the N-half of Sec61 has rotated out of the membrane plane towards the ribosome (Figure 2D). It is striking that the open conformation induced by the TMD is indistinguishable from that induced by an SP (Cα RMSD 0.691 Å; Figure 2C), despite their dissimilar sequences and despite Sec61’s continuous flexibility (Mercier et al., 2021; Itskanov et al., 2021). As an explanation for this bistable behaviour, we observe that the open conformation is stabilised by contacts between the N-half of Sec61α and the ribosome (Figure 2D). The N-half and ribosome have been thought to be isolated from one another, but we find that Sec61 residues 21–27 contact the 28S rRNA helix 24 and uL24, including a particularly well-resolved cation-π interaction between Sec61α R24 and 28S A389. Bacterial SecY lacks this entire loop, perhaps because bacterial secretion is driven by SecA, which competes with ribosomes (Rapoport et al., 2017; Wu et al., 2012; Zimmer et al., 2008). Archaeal SecY, however, does conserve this loop’s structure (Cα RMSD 0.67 Å with Methanocaldococcus jannaschii) and consensus sequence (euk. KPERKIQ vs arc. KPERKVSL, with the cation-π arginine underlined). Stabilising interactions with this widely conserved motif may help Sec61 respond to its diverse substrates with a consistent open state.

Whereas the N-terminal half of RhoTM2 is helical, its C-terminal half loops back through the Sec61 pore in an unfolded conformation similar to the segment of polypeptide downstream of an SP during secretion (Voorhees and Hegde, 2016a; Ma et al., 2019). This is accommodated by the plug helix moving towards the lumenal tip of Sec61γ, effectively becoming part of the channel’s lumenal funnel (Figure 2E), as previously seen with the bacterial plug (Li et al., 2016; Ma et al., 2019). Our observation that different segments of a TMD can simultaneously occupy the lateral gate and central channel is consistent with the through-pore model of insertion, rather than the sliding model (Cymer et al., 2015), at least for this substrate. The through-pore translocation may be favoured because the ribosome exit tunnel’s mouth holds an emerging TMD closer to the Sec61 pore than to the lateral gate. Moreover, the free energy cost of passing a hydrophobic TMD through the hydrophilic pore could be mitigated by the hydrophobic pore ring and partly hydrophilic TMD.

Prior structures of ribosome-bound, open Sec61 disagreed on the structure of the channel pore due to differences in how the rotations of core helices were modelled (Figure 2—figure supplement 1; Braunger et al., 2018; Voorhees and Hegde, 2016a). With ~3.7 Å resolution (Figure 1—figure supplement 2A) and 1.8–3.5 Å predicted aligned error (PAE) restraints from the AlphaFold2 (AF2) prediction of Sec61, we find that the pore is ringed by four aliphatic residues: V85, I183, I292, and L449. Nearby residues F42, I81, I84, L89, I179, I187, I289, and I453 extend hydrophobic patches outwards from the pore ring (Figure 2F). The four pore ring residues identified here are homologous to the four pore ring residues of bacterial SecY (Dalal and Duong, 2009; Ma et al., 2019; Lewis and Hegde, 2021), and adopt similar positions as in the open SecY structure (Cα RMSD 1.1 Å) (Ma et al., 2019). These four residues are pseudosymmetric and likely inherited from SecY’s dimeric ancestor (Lewis and Hegde, 2021). Thus, this structure shows that the likely ancestral pore architecture is universally conserved across the SecY family.

The structure of RhoTM2 trapped during insertion via the central channel of Sec61 represents a paradigm for insertion of N_cyt SAs and the subset of N_cyt TMDs in multipass membrane proteins followed by a long (>100 aa) translocated loop. Both types of substrates are potently inhibited by Sec61 inhibitors (Smalinskaitė et al., 2022; O’Keefe et al., 2021; Zong et al., 2019; McKenna et al., 2017; Morel et al., 2018; Maifeld et al., 2011; Paatero et al., 2016; Tranter et al., 2020) whose binding site at the lateral gate is mutually exclusive with the position of the TMD observed in our structure (Itskanov et al., 2023; Rehan et al., 2023). In the case of N_cyt TMDs followed by a short (<50 aa) translocated loop and another TMD, insertion is thought to occur as a TMD pair via an Oxa1 family member such as the GEL complex within the MPT (Hegde and Keenan, 2022b; Smalinskaitė and Hegde, 2023; Smalinskaitė et al., 2022; Sundaram et al., 2022). Given that Rho biogenesis is not sensitive to Sec61 lateral gate inhibitors, RhoTM2 seems to normally be inserted together with RhoTM3 via the GEL complex (Smalinskaitė et al., 2022). Although the structure seen here probably represents an alternative route captured due to ribosome stalling, it nonetheless proved to be an illuminating paradigm.

RAMP4 occupies the Sec61 gate

Alongside a RhoTM2-bound map, image classification yielded a map in which Sec61 is bound to a different and unexpected density. This density consists of a kinked TMD bound to the Sec61 lateral gate and a ribosome-binding domain (RBD; Figure 3A). The resolution of the RBD density was sufficient for de novo modelling, identifying the sequence as RAMP4. The location, structure, and function of RAMP4 has long been unclear.

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Uncropped full gel images of the immunoblots shown in Figure 3F.

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RAMP4’s RBD is hook-shaped (Figure 3B). Its N-terminus is comprised of an α-helix (residues 5–15) flanked by 3₁₀-helices (3–7,13–20). Subsequent residues then loop back along the helix. The hook is stabilised by an intramolecular hydrogen bond (K13-S28) and a dense network of intermolecular contacts with the ribosome. Specifically, the RBD binds the 28S rRNA’s helices 47, 57, and 59 and ribosomal proteins eL19, 22, and 31, via electrostatic interactions and via hydrophobic interactions with pockets on each of the three ribosomal proteins. The only other factor known to bind in this region is the nascent polypeptide-associated complex subunit β (NACβ) (Jomaa et al., 2022), whose anchor domain has a very different structure but would partly clash with the RAMP4 RBD.

RAMP4’s RBD is connected by a flexible linker to the kinked TMD. This region is less well resolved, so to inform modelling we used AF2 to predict the structure of the Sec61•RAMP4 complex. The resulting prediction is confident (Figure 3—figure supplement 1A) and agrees with the density map. To check that this prediction was specific to RAMP4, we used AF2 to screen a diverse panel of other TMDs and SPs, and found that RAMP4 was indeed the only protein predicted to bind Sec61 (Figure 3—figure supplement 1B). Thus, our model of the RAMP4 TMD is supported by both the density map and a confident, specific structure prediction.

The cytoplasmic half of the RAMP4 TMD is hydrophobic and binds the open Sec61 gate as if it were a TMD or SP (Figure 3C). Bound here, RAMP4 holds the Sec61 pore ring wide, just as SPs and TMDs do (Figure 2C). Unlike an SP or TMD, however, RAMP4 does not displace the plug helix. Instead, the plug moves together with the widening pore ring, keeping it plugged. This observation contrasts with prior speculation that pore widening is sufficient to trigger plug displacement (Voorhees and Hegde, 2016a). Pore widening and unplugging do indeed occur together when the gate is opened by an SP or TMD (Figure 2E), but RAMP4 shows that widening can occur without unplugging. This implies that unplugging occurs when an SP or TMD pulls a flanking segment of nascent chain through the channel pore, and this necessarily displaces the plug. RAMP4, being a tail-anchored protein, threads nothing through the pore, and thus does not clash with the plug.

At its midpoint, the TMD of RAMP4 is kinked 40° at a conserved glycine, and the lumenal half of the TMD is amphipathic. The TMD’s hydrophilic face is oriented towards the channel interior and completes the hydrophilic lumenal funnel of Sec61α (Figure 3D). RAMP4’s integral contribution to forming the Sec61 channel explains why it, and not the more peripheral Sec61β or γ subunits, crosslinks with nascent chain-encoded photoprobes in the Sec61 channel (Schröder et al., 1999).

Comparing different classes of particles, we see that RAMP4 competes with other factors. RAMP4 is partially or completely depleted from classes in which the gate is closed, occupied by RhoTM2, or latched shut by the PAT complex (Figure 3—figure supplement 2). By competing with RAMP4’s TMD, RhoTM2 and PAT also reduce the occupancy of its RBD, which indicates that the RBD alone binds too weakly to be retained during extraction, purification, and grid preparation. This is consistent with biochemical evidence that RAMP4’s association is detergent-sensitive (Görlich and Rapoport, 1993). Together, these factors explain why RAMP4’s occupancy in prior cryo-EM maps was low enough to be overlooked, although in hindsight it is visible in several of them (Braunger et al., 2018; Voorhees et al., 2014; Pfeffer et al., 2015).

To assess the abundance of Sec61•RAMP4 complexes in native membranes, we examined the best native maps currently available, from the subtomogram averages of ribosomes from human cell-derived microsomes (Gemmer et al., 2023b). This dataset was separated into four classes of RTCs: 70% Sec61•TRAP•OSTA, 12% Sec61•MPT, 10% Sec61•TRAP, and 9% Sec61•TRAP•MPT. Examining each class average for the RAMP4 RBD, we find that it is present in the classes without MPT and absent from the classes with MPT, consistent with our single-particle analysis. We then estimated the occupancy of RAMP4 using OccuPy (Forsberg et al., 2023), and found that it is present in ~85% of Sec61•TRAP•OSTA RTCs and ~53% of Sec61•TRAP RTCs (Figure 3—figure supplement 2), which equals ~81% of the non-MPT RTCs. Thus, RAMP4 is absent from MPT-containing RTCs and seems to be present in almost all non-MPT RTCs.

Alongside the RAMP4 RBD, the subtomogram averages also show density for RAMP4’s kinked TMD (Figure 3E). Previously, this TMD density had been speculated to represent SPs (Braunger et al., 2018; Pfeffer et al., 2015; Voorhees and Hegde, 2016b). But the helical density observed is ~25 aa long, like RAMP4’s TMD, whereas the helices of SPs are only 7–15 aa long (von Heijne, 1985). Moreover, the occupancy of this TMD is high, even after hours of translation inhibition (Figure 3—figure supplement 2; Gemmer et al., 2023b), when SP occupancy should be low due to their co-translational dissociation and subsequent processing and degradation (Liaci et al., 2021; Jungnickel and Rapoport, 1995; Mothes et al., 1994; Lyko et al., 1995; Lemberg and Martoglio, 2002). We therefore assign the kinked TMD density in these earlier maps to RAMP4, implying that most co-translational translocation normally occurs through Sec61•RAMP4 channels.

Biochemical analyses provided independent support for the structural observation that RAMP4 and MPT compete for occupancy at the ribosome-Sec61 complex (Figure 3F). In this experiment, HEK293 cells knocked out for either TMCO1 (of the GEL complex) or RAMP4 were engineered to re-express a tagged version at near-native levels. Microsomes from these cells were then used to affinity-purify RTCs via the tagged protein, then immunoblotted for various translocon-associated factors. The results show that RAMP4-purified RTCs contain little or no MPT subcomplexes, but do contain OST and TRAP. By contrast, TMCO1-purified RTCs contain very little RAMP4 and OST, but the full complement of MPT subcomplexes and slightly diminished TRAP. Thus, RAMP4- and MPT-containing RTCs are mostly mutually exclusive, with the former linked to co-translational translocation through the Sec61 channel and the latter linked to membrane protein insertion at the hinge side of a closed Sec61 channel.

Having described the structure of mammalian RAMP4, we briefly consider how widely conserved this structure may be. Examining representative model organisms (S. cerevisiae [Sc] and C. reinhardtii [Cr]), we find that in both cases RAMP4 is confidently predicted to bind Sec61 like animal RAMP4 does (Figure 3—figure supplement 1A). Especially conserved is the region surrounding RAMP4’s glycine kink and Sec61 N300 (Figure 3G and H), which is part of Sec61’s polar cluster that is important for gating (Trueman et al., 2012). To assess when RAMP4 arose in evolution, we searched for homologs in several early-branching eukaryotic taxa (Discoba, Metamonada, Malawimonada) and found it to be largely absent from those groups, suggesting an origin more recent than the last eukaryotic common ancestor. Thus RAMP4, although not ubiquitous, is present across the major eukaryotic kingdoms and forms a dynamically associating part of the Sec61 channel.

uL22’s C-tail switches to contact Sec61 and the nascent chain

In our non-MPT maps, we were surprised to observe density for the C-tail of ribosomal protein uL22 (Figure 4A). This tail has not been described in any prior structural studies. Here, we find it stretched across the ribosome’s membrane-facing surface towards Sec61 and the nascent chain, contacting eL31 and several RNA helices along the way (Figure 4A). Density for the C-terminal helix (CTH) is moderately strong (Figure 1—figure supplement 2A) and similar in each class of particles, indicating that it is not strongly correlated with TRAP or RAMP4 occupancy, nor Sec61 conformation or nascent chain binding to Sec61. The sole exception is the absence of the CTH from the MPT map (Smalinskaitė et al., 2022), where it would clash with the gate latch helices of the PAT complex (Figure 4A). It would also clash with SRP (via SRP54’s M-domain) (Kobayashi et al., 2018; Voorhees and Hegde, 2015; Jomaa et al., 2021), NAC (via its ribosome-binding helices) (Jomaa et al., 2022), RAC (via Zuo1) (Kišonaitė et al., 2023), NatA (via Nat1) (Knorr et al., 2019), and NatB (via MDM20) (Knorr et al., 2023).

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Having seen that uL22 can adopt an engaged or displaced state in different RTCs, we then compared these states to a map of cytoplasmic ribosomes without bound translocons (EMD-40205, the best-resolved mammalian map available) (Holm et al., 2023). The cytoplasmic ribosome displays clear density for the uL22 tail up to the point where it binds H24/47, but the following CTH is diffuse, indicating that it is flexibly anchored to H24/47. Anchored here, the CTH would be well positioned to scan emerging nascent chains and sense when Sec61 binds.

To judge how common uL22 engagement is, we re-examined publicly available RTC maps for previously overlooked uL22 density. We found clear uL22 CTH density in maps from several recent studies (Figure 4—figure supplement 1; Gemmer et al., 2023b; Jaskolowski et al., 2023; Pauwels et al., 2023). Most importantly, it is present in subtomogram averages of RTCs transporting diverse endogenous nascent chains through intact microsomal membranes (Gemmer et al., 2023b), and the uL22 CTH density in those maps is just as strong as in the present dataset (~40% occupancy; Figure 4—figure supplement 1A). This indicates that uL22 engagement is common in native translocons. It is unclear why it is not visible in earlier maps; it may be present and not well resolved, or absent due to a preference for specific nascent chain features.

Comparing the uL22 tail across species, we find that it is conserved by the earliest branches of the animal tree, but not by the nearest non-animal branches (Figure 4B). Although fungi have an elongated uL22 tail, it is unlike the animal tail in sequence, and appears to have arisen independently. Indeed structures show that the fungal uL22 tail binds to an entirely different site (Figure 4C; Best et al., 2023), and does so constitutively instead of dynamically. Taken together, this suggests that the animal uL22 tail structure was acquired during the evolution of the first animals. Among animals, the uL22 tail’s conservation suggests that it is functionally important. Particularly well conserved is the SXKK motif that initiates the CTH and binds H24/47 (Figure 4D). The tip of the CTH that would contact nascent chains typically contains a mix of basic, acidic, and hydrophobic residues, and this complexity makes it difficult to predict what if any nascent chain features it may recognise.

It is noteworthy that when engaged, the uL22 CTH blocks a gap between the ribosome and Sec61 that would otherwise allow the nascent chain to exit the channel vestibule and enter the cytoplasm (Figure 4E). This gate-side exit is one of two such exits, with the other being on the Sec61 hinge side, where multipass proteins exit for insertion by the MPT (Smalinskaitė et al., 2022). While uL22 blocks the gate-side exit, the hinge-side exit would remain open. The functional consequences of this change are currently unclear.

Structure of the TRAP complex

The occupancy of the hetero-tetrameric TRAP complex (comprised of α, β, γ, and δ subunits) in the all-particle map was low, so we performed focused classification on this region and obtained two well-resolved TRAP classes (Figure 1—figure supplement 1C). TRAP in these two classes adopts slightly different tilts with respect to the ribosome, which we call conformations 1 and 2, although the true range of TRAP conformations is presumably continuous rather than discrete. The TRAP maps were well fit by a predicted model (Figure 5A) after minor adjustments. One additional protein density was observed in TRAP class 1, consisting of a TMD and cytoplasmic helix bound to TRAPγ. We provisionally assign this density to Calnexin because it is by far the major TRAP-binding protein (Fons et al., 2003; Wada et al., 1991) and it is known to bind TRAP via its TMD. The putative TRAP-Calnexin interaction was not detected by AF2, perhaps because Calnexin di-palmitoylation, which AF2 ignores, is crucial for TRAP interaction with Calnexin (Lakkaraju et al., 2012). Anchoring of Calnexin at this position would explain how its elongated lumenal domain interacts co-translationally with nascent chains as they are translocated by RTCs (Molinari and Helenius, 2000; Daniels et al., 2003).

Figure 5

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The TMDs of TRAPβγδ and the provisionally assigned Calnexin all bundle together, but not the TMD of TRAPα. Instead, the TRAPα TMD binds the lumenal hinge loop of Sec61α, as does the TRAPα lumenal domain (Figure 5B.3). While making those lumenal contacts, the TRAPα TMD tilts its cytoplasmic end away from Sec61α, allowing conserved basic residues flanking its TMD to contact the tip of 5.8S rRNA helix 7 (Figure 5B.4). Strikingly, this rRNA segment has undergone a dramatic rearrangement from its cytoplasmic state, which puts C81, U85, and U86 in contact with the membrane and the basic residues in TRAP (Figure 5B.5). From there, the C-tail of TRAPα continues along the ribosome surface and binds tightly at a site contacting uL23, uL29, and 5.8S rRNA helix 9 (Figure 5B.6). Thus TRAPα makes extensive contacts with both the ribosome and Sec61.

Besides TRAPα, TRAPβ, TRAPγ, and Calnexin also contact the ribosome and Sec61: TRAPβ’s basic C-terminal residues contact the 5.8S rRNA at H9 ES3 (Figure 5B.7); TRAPγ’s helical hairpin residues R110, K111, and especially R114 contact the 28S rRNA at H54 and ES26 (Figure 5B.8); TRAPγ’s flexible N-tail helix binds eL38 (Figure 5B.9); the provisionally assigned Calnexin’s flexible C-tail contacts the 28S rRNA at H63 ES27a (Figure 5B.10); TRAPγ’s C-terminus contacts Sec61γ’s N-terminus (Figure 5B.1); and TRAPγ’s lumenal loop 3/4 contacts Sec61α’s lumenal loop 8/9 (Figure 5B.2). Altogether, TRAP’s multivalent site-specific and non-specific interactions across many sites via both rigid and flexible elements explain how it remains associated with the translocon while adopting a range of positions and orientations, compared to other translocation factors like OST or the MPT which bind at relatively fixed sites.

TRAP competes and cooperates with different translocon subunits

The TRAPδ Ig-like domain contains a helical hairpin between β-strands 6 and 7. No similar hairpin appears in any other domain in the AFDB50 database queried by Foldseek (van Kempen et al., 2024; Figure 6—figure supplement 1A). This unique hairpin is isolated in our Sec61-TRAP structure, but in Sec61-TRAP-OSTA structures (Gemmer et al., 2023b), it extends towards OST-A subunit RPN2 (Figure 6A). A basic patch on TRAPδ is separated by less than 5 Å from an acidic patch on RPN2, indicating that they would share an electrostatic attraction (Figure 6A, inset). Each partner presents three conserved charges, and the closest pair (K117 on TRAPδ and D386 on RPN2) is the most conserved, indicating that their attraction is functionally important. OSTA’s attraction to TRAPδ is weak compared to its binding to the ribosome, but TRAPδ may nonetheless help recruit OSTA, since TRAPδ would attract OSTA from most possible angles of approach, whereas OSTA’s ribosome contacts are stereospecific. The TRAPδ-OSTA interaction may explain why TRAPδ defects cause congenital disorders of glycosylation (Losfeld et al., 2014; Ng et al., 2019; Phoomak et al., 2021).

Figure 6 with 1 supplement see all

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Comparing the TRAP structure to the MPT structure (Smalinskaitė et al., 2022), we find that TRAP would clash with part of the MPT, namely the BOS complex comprised of TMEM147, Nicalin, and NOMO. The prior BOS model omitted NOMO, so to fully characterise this clash we ran a prediction of the full BOS complex structure, obtaining a high-confidence prediction that fits a previously reported subtomogram average (Braunger et al., 2018; Figure 6B). As an aside, it is noteworthy that the first prealbumin-like domain (PLD1) in NOMO is predicted to bind PLD10 and PLD11, suggesting that its reported ability to regulate the spacing between ER sheets (Amaya et al., 2021) results from it forming antiparallel homodimers across the ER lumen (Figure 6—figure supplement 1B). Contemporaneous work (Gemmer et al., 2023a) has arrived at a similar model for PLD10-12 but did not model PLD1.

Comparing the TRAP and BOS structures, TRAPα competes with BOS subunit TMEM147 for the same binding sites on the hinge loop of Sec61α and the 5.8S rRNA’s helix 7, while PLD12 of NOMO would clash to a more limited degree with the lumenal domain of TRAPβ. When TRAP is displaced by BOS, its interactions with Sec61 are disrupted and its transmembrane bundle (which is no longer visible in the cryo-EM map) evidently pivots towards the still-bound TRAPα C-tail RBD, causing a pronounced shift in the shape of the detergent micelle (Figure 6C). This extensive competition resulting in partial TRAP displacement explains why prior studies suggested that TRAP is present in only 40% of MPT complexes, but at high occupancy at all other RTCs (Gemmer et al., 2023b; Gemmer et al., 2023a). However, the high occupancy of the TRAPα C-tail RBD in MPT-containing translocons suggests that TRAP does not fully dissociate upon MPT recruitment and remains in vicinity of the translocation site.

Additional functionally relevant TRAP features

The above analysis shows that TRAP binds together ribosomes, OSTA, and Sec61, but competes with the MPT, whose presence inhibits Sec61. In principle, these activities could suffice to explain TRAP’s observed functions as a stimulator of Sec61-dependent secretion (Fons et al., 2003) and OSTA-dependent glycosylation (Phoomak et al., 2021). However, we observe four additional TRAP features that appear functionally relevant and are therefore noteworthy.

First, TRAPα presents a conserved patch to the nascent chain where it first emerges from Sec61 (Figure 7A). This patch could potentially bind the nascent chain or redirect it towards the active site of OSTA. Second, by binding the C-half of Sec61, TRAP can potentially influence the opening of the lateral gate (Figure 7B). If TRAP caused the C-half to favour opening, this would explain in part why it stimulates the recognition of weakly gating signals (Fons et al., 2003). The combined effect could explain its overall stimulatory role in translocation.

Figure 7

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Third, TRAP prefers a membrane plane tilted 20° relative to Sec61’s, and imposes this curvature on the surrounding micelle (Figure 7C). The same tilt is observed in Sec61-TRAP maps from intact membranes, but it is unclear whether TRAP imposed this curvature on the membrane, since it is the same degree of curvature found in native ER tubes and sheet edges (Shibata et al., 2010). Thus, while it is possible that TRAP modulates Sec61 activity by disrupting the local membrane, it may instead have adapted to reside in and sense pre-existing membrane deformations.

Fourth, the lumenal domain of TRAPα near the Sec61 channel has an extraordinarily long and acidic N-tail tipped by hydrophobic residues (Figure 7D). This could interact with any lumenal part of the translocon or nascent chain. No data is available on this tail’s function, aside from the fact that this and Calnexin each bind far more calcium than any other protein in ER membrane extracts (Wada et al., 1991) as expected for its exceptional charge and abundance.

TRAP conservation across eukaryotes and archaea

Having described the features of metazoan TRAP, here we briefly contrast it with TRAP in representative model organisms from plantae (Cr), fungi (Sc), and excavates (Trypanosoma brucei [Tb]), a group of early-branching eukaryotes. Cr has already been shown by subtomogram averaging to contain TRAPαβ-like density at its translocons (Pfeffer et al., 2017). Indeed we find that its genome contains TRAPαβ but not γδ, and the predicted structure of Cr TRAPαβ-Sec61 fits the tomographic density with minor adjustments (Figure 8A). Cr TRAP conserves animal TRAP’s predicted binding to the Sec61 hinge and its C-tail RBD. However, whereas animal TRAP binds to the ribosome at eL38 via TRAPγ, Cr TRAP is predicted to bind this same ribosomal protein via TRAPβ (Figure 8A, inset). Thus, the Cr and animal complexes share the similar ribosome-binding sites despite their differences in composition. As an aside, it is noteworthy that the Cr TRAPβ TMD is too hydrophilic to insert on its own (∆G_pred = 1.824, i.e., 5% insertion), suggesting that it is bound and stabilised by an unidentified protein functionally analogous to TRAPγ.

Figure 8

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Sc has no annotated TRAP genes, and indeed it has been suggested that most fungi lack TRAP (Pfeffer et al., 2017). However, we find that Sc contains a previously unidentified gene for TRAPα (Irc22, HHpred E=10^–35). This suggests TRAP’s prevalence in fungi has been underestimated. Sc TRAPα conserves animal TRAPα’s predicted binding to the Sec61 hinge and its C-tail RBD (Figure 8B). No other TRAP subunits were detected in cerevisiae by sequence- or structure-based searches (HHpred, Foldseek). Thus TRAP’s links to co-translational translocation may be conserved in fungi, but its complexity is dramatically reduced.

The early-branching excavate Tb has TRAPα, β, and γ, but no δ (Figure 8C). Tb TRAPαγ conserve the same predicted interactions as their homologs in fungi, plants, and animals (where found). Overall, the observed distribution and conservation of features suggest that the original eukaryotic TRAP consisted of TRAPαβγ, bound the Sec61 hinge loop via TRAPα, bound the ribosome flexibly via the TRAPα C-tail RBD and the TRAPγ N-tail, and also contacted the ribosome via the TRAPβγ bundle.

If TRAPαβγ were present in the last eukaryotic common ancestor, they may have been inherited from archaea, which are the ancestors of eukaryotes. Structural queries of eukaryotes’ archaeal sister group (Heimdallarchaeota) identified candidate homologs of TRAPα (E=9.95 × 10^–8), β (E=5.15 × 10^–10), possibly TRAPγ (E=1.84), and not TRAPδ, as expected. To test whether these candidates are also similar to TRAPαβγ in sequence, we used them to perform reciprocal HHpred queries of the human proteome, and in each case the corresponding human TRAP protein was the top hit (E=0.031 for TRAPα, 9.4×10^–14 for TRAP β, and 110 for TRAPγ). A contemporaneous study has also proposed TRAP homologs in Heimdallarchaeota (Eme et al., 2023), although caution is warranted in some of these assignments because many do not share predicted structural similarity to TRAP subunits or sequence similarity with human TRAP in reciprocal HHpred searches.

We then queried AF2 to see whether the top-scoring archaeal TRAPαβγ hits were predicted to form a complex with each other or with SecY. No contacts among the top hits were detected, but this could be an artefact of how few sequences AF2 could collect to constrain the prediction (as few as 5). By contrast, contacts were confidently predicted between archaeal TRAPα and SecY, forming a dimer similar to the eukaryotic TRAPα-Sec61 dimer (Figure 8D). This similarity was predicted even though no eukaryotic TRAPα were included in the input alignment or training set, and thus it was based on archaeal TRAPα alone.

While performing sequence searches with TRAPα and β, we were surprised to find that they are much more similar in sequence than we had expected (HHpred p=2 × 10^–7), suggesting that they share a common ancestor. In fact they are more similar to one another than to any other human proteins (HHpred), including other Ig-like domains. By contrast, TRAPδ is quite dissimilar (HHpred α p=0.64, β 0.66). Thus, although TRAP’s three lumenal domains all have Ig-like folds, it appears that two of them, TRAPα and β, originated when an archaeal proto-TRAP protein duplicated, whereas TRAPδ is evolutionarily unrelated (Figure 8E). If proto-TRAP formed dimers, it would presumably resemble a homomeric version of the heterodimeric TRAPαβ complex found in plants (Pfeffer et al., 2017).

Towards understanding TRAP’s mechanism, it is noteworthy that although its affinity for the ribosome and Sec61 are conserved, at least two of the four other functionally relevant features highlighted above are not conserved: yeast TRAP’s single TMD is unlikely to deform membranes, and TRAP from many organisms does not have a polyacidic lumenal domain. The other two features, allosteric effects on gating and a putative substrate-binding activity, could plausibly be conserved together with the TRAPα-Sec61 structure.

The EM maps have been deposited in EMDB with the accession numbers EMD-19195, EMD-19196, EMD-19197, EMD-19198, 19199, EMD-19200, EMD-19201, EMD-19202, EMD-19203, and EMD-19204. The molecular models have been deposited in the PDB with accession numbers 8RJB, 8RJC, and 8RJD. The AlphaFold2-predicted structures (Varadi et al., 2024) reported in this study have been deposited in ModelArchive with accession numbers ma-jjnuw, ma-hbsof, ma-hknzh, ma-x3uvj, ma-j8wag, ma-9gsmk, ma-l9qqq, and ma-ise4t.

1. 1. Lewis AJO
   2. Hegde RS

   (2024) Electron Microscopy Data Bank

   ID EMD-19195. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61-RAMP4.

   https://www.ebi.ac.uk/emdb/EMD-19195
2. 1. Lewis AJO
   2. Hegde RS

   (2024) Electron Microscopy Data Bank

   ID EMD-19196. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61, all-particle reconstruction.

   https://www.ebi.ac.uk/emdb/EMD-19196
3. 1. Lewis AJO
   2. Hegde RS

   (2024) Electron Microscopy Data Bank

   ID EMD-19197. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61-TRAP, open conformation 1.

   https://www.ebi.ac.uk/emdb/EMD-19197
4. 1. Lewis AJO
   2. Hegde RS

   (2024) Electron Microscopy Data Bank

   ID EMD-19198. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61-TRAP, open conformation 2.

   https://www.ebi.ac.uk/emdb/EMD-19198
5. 1. Lewis AJO
   2. Hegde RS

   (2024) Electron Microscopy Data Bank

   ID EMD-19199. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61, bound TRAP-alpha TMD.

   https://www.ebi.ac.uk/emdb/EMD-19199
6. 1. Lewis AJO
   2. Hegde RS

   (2024) Electron Microscopy Data Bank

   ID EMD-19200. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61-TRAP, conformation 1.

   https://www.ebi.ac.uk/emdb/EMD-19200
7. 1. Lewis AJO
   2. Hegde RS

   (2024) Electron Microscopy Data Bank

   ID EMD-19201. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61-TRAP, conformation 2.

   https://www.ebi.ac.uk/emdb/EMD-19201
8. 1. Lewis AJO
   2. Hegde RS

   (2024) Electron Microscopy Data Bank

   ID EMD-19202. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61, closed conformation 1.

   https://www.ebi.ac.uk/emdb/EMD-19202
9. 1. Lewis AJO
   2. Hegde RS

   (2024) Electron Microscopy Data Bank

   ID EMD-19203. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61, closed conformation 2.

   https://www.ebi.ac.uk/emdb/EMD-19203
10. 1. Lewis AJO
    2. Hegde RS

    (2024) Electron Microscopy Data Bank

    ID EMD-19204. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Rho-bound Sec61.

    https://www.ebi.ac.uk/emdb/EMD-19204
11. 1. Lewis AJO
    2. Hegde RS

    (2024) RCSB Protein Data Bank

    ID 8RJB. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61-RAMP4.

    https://www.rcsb.org/structure/8RJB
12. 1. Lewis AJO
    2. Hegde RS

    (2024) RCSB Protein Data Bank

    ID 8RJC. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61-TRAP, open conformation 1.

    https://www.rcsb.org/structure/8RJC
13. 1. Lewis AJO
    2. Hegde RS

    (2024) RCSB Protein Data Bank

    ID 8RJD. Structure of the rabbit 80S ribosome stalled on a 2-TMD rhodopsin intermediate in complex with Sec61-TRAP, open conformation 2.

    https://www.rcsb.org/structure/8RJD
14. 1. Lewis AJO
    2. Hegde RS

    (2024) ModelArchive

    ID ma-jjnuw. Chlamydomonas reinhardtii Sec61-RAMP4 complex.

    https://modelarchive.org/doi/10.5452/ma-jjnuw
15. 1. Lewis AJO
    2. Hedge RS

    (2024) ModelArchive

    ID ma-hbsof. Saccharomyces cerevisiae Sec61-RAMP4 complex (aka Ysy6p).

    https://modelarchive.org/doi/10.5452/ma-hbsof
16. 1. Lewis AJO
    2. Hegde RS

    (2024) ModelArchive

    ID ma-hknzh. Canis lupus familiaris Sec61-RAMP4 complex.

    https://modelarchive.org/doi/10.5452/ma-hknzh
17. 1. Lewis AJO
    2. Hegde RS

    (2024) ModelArchive

    ID ma-x3uvj. Heimdallarchaean TRAPα-SecY complex.

    https://modelarchive.org/doi/10.5452/ma-x3uvj
18. 1. Lewis AJO
    2. Hegde RS

    (2024) ModelArchive

    ID ma-j8wag. Trypanosoma brucei TRAP-Sec61 complex.

    https://modelarchive.org/doi/10.5452/ma-j8wag
19. 1. Lewis AJO
    2. Hegde RS

    (2024) ModelArchive

    ID ma-9gsmk. Chlamydomonas reinhardtii TRAP-Sec61-RAMP4-eL38 complex.

    https://modelarchive.org/doi/10.5452/ma-9gsmk
20. 1. Lewis AJO
    2. Hegde RS

    (2024) ModelArchive

    ID ma-l9qqq. Saccharomyces cerevisiae Sec61-TRAP complex (aka Irc22p).

    https://modelarchive.org/doi/10.5452/ma-l9qqq
21. 1. Lewis AJO
    2. Hegde RS

    (2024) ModelArchive

    ID ma-ise4t. Canis lupus familiaris BOS complex (TMEM147/Nicalin/NOMO, C. lupus familiaris).

    https://modelarchive.org/doi/10.5452/ma-ise4t

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   (2023) RCSB Protein Data Bank

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   https://www.rcsb.org/structure/8AGX
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   (2023) RCSB Protein Data Bank

   ID 8B2L. Structure of the actively translating plant 80S ribosome at 2.2 Å resolution.

   https://www.rcsb.org/structure/8B2L
3. 1. Braunger K
   2. Pfeffer S
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   (2018) RCSB Protein Data Bank

   ID 6FTI. Structural basis for coupling protein transport and N-glycosylation at the mammalian endoplasmic reticulum.

   https://www.rcsb.org/structure/6FTI
4. 1. Holm M
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   (2023) RCSB Protein Data Bank

   ID 8GLP. mRNA decoding in human is kinetically and structurally distinct from bacteria.

   https://www.rcsb.org/structure/8GLP
5. 1. Holm M
   2. Natchiar SK
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   4. Myasnikov AG
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   ID EMD-40205. mRNA decoding in human is kinetically and structurally distinct from bacteria.

   https://www.ebi.ac.uk/emdb/EMD-40205
6. 1. Pfeffer S
   2. Dudek J
   3. Schaffer M
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   ID EMD-4145. Dissecting the molecular organization of the translocon-associated protein complex.

   https://www.ebi.ac.uk/emdb/EMD-4145
7. 1. Braunger K
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   7. Becker T
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   (2018) EMDB

   ID EMD-4311. Structural basis for coupling protein transport and N-glycosylation at the mammalian endoplasmic reticulum.

   https://www.ebi.ac.uk/emdb/EMD-4311
8. 1. Voorhees RM
   2. Hegde RS

   (2016) RCSB Protein Data Bank

   ID 3JC2. The structure of the mammalian Sec61 channel opened by a signal sequence.

   https://www.rcsb.org/structure/3JC2
9. 1. Voorhees RM
   2. Hegde RS

   (2016) EMDB

   ID EMD-3245. The structure of the mammalian Sec61 channel opened by a signal sequence.

   https://www.ebi.ac.uk/emdb/EMD-3245
10. 1. Smalinskaitė L
    2. Kim MK
    3. Lewis AJO
    4. Keenan RJ
    5. Hegde RS

    (2022) RCSB Protein Data Bank

    ID 7TM3. Mechanism of an intramembrane chaperone for multipass membrane proteins.

    https://www.rcsb.org/structure/7TM3
11. 1. Smalinskaite L
    2. Kim MK
    3. Lewis AJO
    4. Keenan RJ
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    (2022) EMDB

    ID EMD-25994. Mechanism of an intramembrane chaperone for multipass membrane proteins.

    https://www.ebi.ac.uk/emdb/EMD-25994
12. 1. Gemmer M
    2. Chaillet ML
    3. van Loenhout J

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    ID EMD-15885. Visualization of translation and protein biogenesis at the ER membrane.

    https://www.ebi.ac.uk/emdb/EMD-15885
13. 1. Gemmer M
    2. Chaillet ML
    3. van Loenhout J

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    ID EMD-15889. Visualization of translation and protein biogenesis at the ER membrane.

    https://www.ebi.ac.uk/emdb/EMD-15889
14. 1. Gemmer M
    2. Fedry JMM
    3. Forster FG

    (2022) RCSB Protein Data Bank

    ID 8B6L. Subtomogram average of the human Sec61-TRAP-OSTA-translocon.

    https://www.rcsb.org/structure/8B6L
15. 1. Gemmer M
    2. Fedry JMM
    3. Forster FG

    (2022) EMDB

    ID EMD-15870. Subtomogram average of the human Sec61-TRAP-OSTA-translocon.

    https://www.ebi.ac.uk/emdb/EMD-15870

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Author details

1. Aaron JO Lewis

   MRC Laboratory of Molecular Biology, Cambridge, United Kingdom

   Contribution

   Conceptualization, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8818-1763

2. Frank Zhong

   Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

3. Robert J Keenan

   Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, United States

   Contribution

   Supervision, Funding acquisition, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1466-0889

4. Ramanujan S Hegde

   MRC Laboratory of Molecular Biology, Cambridge, United Kingdom

   Contribution

   Conceptualization, Supervision, Project administration, Writing – review and editing

   For correspondence

   rhegde@mrc-lmb.cam.ac.uk

   Competing interests

   Scientific advisor and equity holder of Gate Bioscience

   "This ORCID iD identifies the author of this article:" 0000-0001-8338-852X

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