Structural analysis of the dynamic ribosome-translocon complex.

The protein translocon at the endoplasmic reticulum comprises the Sec61 translocation channel and numerous accessory factors that collectively facilitate the biogenesis of secretory and membrane proteins. Here, we leveraged recent advances in cryo-electron microscopy (cryo-EM) and structure prediction to derive insights into several novel configurations of the ribosome-translocon complex. We show how a transmembrane domain (TMD) in a looped configuration passes through the Sec61 lateral gate during membrane insertion; how a nascent chain can bind and constrain the conformation of ribosomal protein uL22; and how the translocon-associated protein (TRAP) complex can adjust its position during different stages of protein biogenesis. Most unexpectedly, we find that a large proportion of translocon complexes contains RAMP4 intercalated into Sec61's lateral gate, widening Sec61's central pore and contributing to its hydrophilic interior. These structures lead to mechanistic hypotheses for translocon function and highlight a remarkably plastic machinery whose conformations and composition adjust dynamically to its diverse range of substrates.

High-confidence 3D template matching for cryo-electron tomography.

Visual proteomics attempts to build atlases of the molecular content of cells but the automated annotation of cryo electron tomograms remains challenging. Template matching (TM) and methods based on machine learning detect structural signatures of macromolecules. However, their applicability remains limited in terms of both the abundance and size of the molecular targets. Here we show that the performance of TM is greatly improved by using template-specific search parameter optimization and by including higher-resolution information. We establish a TM pipeline with systematically tuned parameters for the automated, objective and comprehensive identification of structures with confidence 10 to 100-fold above the noise level. We demonstrate high-fidelity and high-confidence localizations of nuclear pore complexes, vaults, ribosomes, proteasomes, fatty acid synthases, lipid membranes and microtubules, and individual subunits inside crowded eukaryotic cells. We provide software tools for the generic implementation of our method that is broadly applicable towards realizing visual proteomics.

What shapes template-matching performance in cryogenic electron tomography in situ?

The detection of specific biological macromolecules in cryogenic electron tomography data is frequently approached by applying cross-correlation-based 3D template matching. To reduce computational cost and noise, high binning is used to aggregate voxels before template matching. This remains a prevalent practice in both practical applications and methods development. Here, the relation between template size, shape and angular sampling is systematically evaluated to identify ribosomes in a ground-truth annotated data set. It is shown that at the commonly used binning, a detailed subtomogram average, a sphere and a heart emoji result in near-identical performance. These findings indicate that with current template-matching practices macromolecules can only be detected with high precision if their shape and size are sufficiently different from the background. Using theoretical considerations, the experimental results are rationalized and it is discussed why primarily low-frequency information remains at high binning and that template matching fails to be accurate because similarly shaped and sized macromolecules have similar low-frequency spectra. These challenges are discussed and potential enhancements for future template-matching methodologies are proposed.

Structural mechanism of angiogenin activation by the ribosome.

Angiogenin, an RNase-A-family protein, promotes angiogenesis and has been implicated in cancer, neurodegenerative diseases and epigenetic inheritance1-10. After activation during cellular stress, angiogenin cleaves tRNAs at the anticodon loop, resulting in translation repression11-15. However, the catalytic activity of isolated angiogenin is very low, and the mechanisms of the enzyme activation and tRNA specificity have remained a puzzle3,16-23. Here we identify these mechanisms using biochemical assays and cryogenic electron microscopy (cryo-EM). Our study reveals that the cytosolic ribosome is the activator of angiogenin. A cryo-EM structure features angiogenin bound in the A site of the 80S ribosome. The C-terminal tail of angiogenin is rearranged by interactions with the ribosome to activate the RNase catalytic centre, making the enzyme several orders of magnitude more efficient in tRNA cleavage. Additional 80S-angiogenin structures capture how tRNA substrate is directed by the ribosome into angiogenin's active site, demonstrating that the ribosome acts as the specificity factor. Our findings therefore suggest that angiogenin is activated by ribosomes with a vacant A site, the abundance of which increases during cellular stress24-27. These results may facilitate the development of therapeutics to treat cancer and neurodegenerative diseases.

Structures of the ribosome bound to EF-Tu-isoleucine tRNA elucidate the mechanism of AUG avoidance.

The frequency of errors upon decoding of messenger RNA by the bacterial ribosome is low, with one misreading event per 1 × 104 codons. In the universal genetic code, the AUN codon box specifies two amino acids, isoleucine and methionine. In bacteria and archaea, decoding specificity of the AUA and AUG codons relies on the wobble avoidance strategy that requires modification of C34 in the anticodon loop of isoleucine transfer RNAIleCAU (tRNAIleCAU). Bacterial tRNAIleCAU with 2-lysylcytidine (lysidine) at the wobble position deciphers AUA while avoiding AUG. Here we report cryo-electron microscopy structures of the Escherichia coli 70S ribosome complexed with elongation factor thermo unstable (EF-Tu) and isoleucine-tRNAIleLAU in the process of decoding AUA and AUG. Lysidine in tRNAIleLAU excludes AUG by promoting the formation of an unusual Hoogsteen purine-pyrimidine nucleobase geometry at the third position of the codon, weakening the interactions with the mRNA and destabilizing the EF-Tu ternary complex. Our findings elucidate the molecular mechanism by which tRNAIleLAU specifically decodes AUA over AUG.

A new family of bacterial ribosome hibernation factors.

To conserve energy during starvation and stress, many organisms use hibernation factor proteins to inhibit protein synthesis and protect their ribosomes from damage1,2. In bacteria, two families of hibernation factors have been described, but the low conservation of these proteins and the huge diversity of species, habitats and environmental stressors have confounded their discovery3-6. Here, by combining cryogenic electron microscopy, genetics and biochemistry, we identify Balon, a new hibernation factor in the cold-adapted bacterium Psychrobacter urativorans. We show that Balon is a distant homologue of the archaeo-eukaryotic translation factor aeRF1 and is found in 20% of representative bacteria. During cold shock or stationary phase, Balon occupies the ribosomal A site in both vacant and actively translating ribosomes in complex with EF-Tu, highlighting an unexpected role for EF-Tu in the cellular stress response. Unlike typical A-site substrates, Balon binds to ribosomes in an mRNA-independent manner, initiating a new mode of ribosome hibernation that can commence while ribosomes are still engaged in protein synthesis. Our work suggests that Balon-EF-Tu-regulated ribosome hibernation is a ubiquitous bacterial stress-response mechanism, and we demonstrate that putative Balon homologues in Mycobacteria bind to ribosomes in a similar fashion. This finding calls for a revision of the current model of ribosome hibernation inferred from common model organisms and holds numerous implications for how we understand and study ribosome hibernation.

mRNA reading frame maintenance during eukaryotic ribosome translocation.

One of the most critical steps of protein synthesis is coupled translocation of messenger RNA (mRNA) and transfer RNAs (tRNAs) required to advance the mRNA reading frame by one codon. In eukaryotes, translocation is accelerated and its fidelity is maintained by elongation factor 2 (eEF2)1,2. At present, only a few snapshots of eukaryotic ribosome translocation have been reported3-5. Here we report ten high-resolution cryogenic-electron microscopy (cryo-EM) structures of the elongating eukaryotic ribosome bound to the full translocation module consisting of mRNA, peptidyl-tRNA and deacylated tRNA, seven of which also contained ribosome-bound, naturally modified eEF2. This study recapitulates mRNA-tRNA2-growing peptide module progression through the ribosome, from the earliest states of eEF2 translocase accommodation until the very late stages of the process, and shows an intricate network of interactions preventing the slippage of the translational reading frame. We demonstrate how the accuracy of eukaryotic translocation relies on eukaryote-specific elements of the 80S ribosome, eEF2 and tRNAs. Our findings shed light on the mechanism of translation arrest by the anti-fungal eEF2-binding inhibitor, sordarin. We also propose that the sterically constrained environment imposed by diphthamide, a conserved eukaryotic posttranslational modification in eEF2, not only stabilizes correct Watson-Crick codon-anticodon interactions but may also uncover erroneous peptidyl-tRNA, and therefore contribute to higher accuracy of protein synthesis in eukaryotes.

Visualizing translation dynamics at atomic detail inside a bacterial cell.

Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells1. Here we use advances in cryo-electron tomography and sub-tomogram analysis2,3 to visualize the structural dynamics of translation inside the bacterium Mycoplasma pneumoniae. To interpret the functional states in detail, we first obtain a high-resolution in-cell average map of all translating ribosomes and build an atomic model for the M. pneumoniae ribosome that reveals distinct extensions of ribosomal proteins. Classification then resolves 13 ribosome states that differ in their conformation and composition. These recapitulate major states that were previously resolved in vitro, and reflect intermediates during active translation. On the basis of these states, we animate translation elongation inside native cells and show how antibiotics reshape the cellular translation landscapes. During translation elongation, ribosomes often assemble in defined three-dimensional arrangements to form polysomes4. By mapping the intracellular organization of translating ribosomes, we show that their association into polysomes involves a local coordination mechanism that is mediated by the ribosomal protein L9. We propose that an extended conformation of L9 within polysomes mitigates collisions to facilitate translation fidelity. Our work thus demonstrates the feasibility of visualizing molecular processes at atomic detail inside cells.

Uncovering translation roadblocks during the development of a synthetic tRNA.

Ribosomes are remarkable in their malleability to accept diverse aminoacyl-tRNA substrates from both the same organism and other organisms or domains of life. This is a critical feature of the ribosome that allows the use of orthogonal translation systems for genetic code expansion. Optimization of these orthogonal translation systems generally involves focusing on the compatibility of the tRNA, aminoacyl-tRNA synthetase, and a non-canonical amino acid with each other. As we expand the diversity of tRNAs used to include non-canonical structures, the question arises as to the tRNA suitability on the ribosome. Specifically, we investigated the ribosomal translation of allo-tRNAUTu1, a uniquely shaped (9/3) tRNA exploited for site-specific selenocysteine insertion, using single-molecule fluorescence. With this technique we identified ribosomal disassembly occurring from translocation of allo-tRNAUTu1 from the A to the P site. Using cryo-EM to capture the tRNA on the ribosome, we pinpointed a distinct tertiary interaction preventing fluid translocation. Through a single nucleotide mutation, we disrupted this tertiary interaction and relieved the translation roadblock. With the continued diversification of genetic code expansion, our work highlights a targeted approach to optimize translation by distinct tRNAs as they move through the ribosome.

Continued expansion of the genetic code has required the use of synthetic tRNAs for decoding. Some of these synthetic tRNAs have unique structural features that are not observed in canonical tRNAs. Here, the authors applied single-molecule, biochemical and structural methods to determine whether these distinct features were deleterious for efficient protein translation on the ribosome. With a focus on selenocysteine insertion, the authors explored an allo-tRNA with a 9/3 acceptor domain. They observed a translational roadblock that occurred in A to P site tRNA translocation. This block was mediated by a tertiary interaction across the tRNA core, directing the variable arm position into an unfavorable conformation. A single-nucleotide mutation disrupted this interaction, providing flexibility in the variable arm and promoting efficient protein production.

Cryo-EM structure and rRNA modification sites of a plant ribosome.

Protein synthesis in crop plants contributes to the balance of food and fuel on our planet, which influences human metabolic activity and lifespan. Protein synthesis can be regulated with respect to changing environmental cues via the deposition of chemical modifications into rRNA. Here, we present the structure of a plant ribosome from tomato and a quantitative mass spectrometry analysis of its rRNAs. The study reveals fine features of the ribosomal proteins and 71 plant-specific rRNA modifications, and it re-annotates 30 rRNA residues in the available sequence. At the protein level, isoAsp is found in position 137 of uS11, and a zinc finger previously believed to be universal is missing from eL34, suggesting a lower effect of zinc deficiency on protein synthesis in plants. At the rRNA level, the plant ribosome differs markedly from its human counterpart with respect to the spatial distribution of modifications. Thus, it represents an additional layer of gene expression regulation, highlighting the molecular signature of a plant ribosome. The results provide a reference model of a plant ribosome for structural studies and an accurate marker for molecular ecology.

CryoEM structures of pseudouridine-free ribosome suggest impacts of chemical modifications on ribosome conformations.

Pseudouridine, the most abundant form of RNA modification, is known to play important roles in ribosome function. Mutations in human DKC1, the pseudouridine synthase responsible for catalyzing the ribosome RNA modification, cause translation deficiencies and are associated with a complex cancer predisposition. The structural basis for how pseudouridine impacts ribosome function remains uncharacterized. Here, we characterized structures and conformations of a fully modified and a pseudouridine-free ribosome from Saccharomyces cerevisiae in the absence of ligands or when bound with translocation inhibitor cycloheximide by electron cryomicroscopy. In the modified ribosome, the rearranged N1 atom of pseudouridine is observed to stabilize key functional motifs by establishing predominately water-mediated close contacts with the phosphate backbone. The pseudouridine-free ribosome, however, is devoid of such interactions and displays conformations reflective of abnormal inter-subunit movements. The erroneous motions of the pseudouridine-free ribosome may explain its observed deficiencies in translation.

Annealing synchronizes the 70S ribosome into a minimum-energy conformation.

Researchers commonly anneal metals, alloys, and semiconductors to repair defects and improve microstructures via recrystallization. Theoretical studies indicate that simulated annealing on biological macromolecules helps predict the final structures with minimum free energy. Experimental validation of this homogenizing effect and further exploration of its applications are fascinating scientific questions that remain elusive. Here, we chose the apo-state 70S ribosome from Escherichia coli as a model, wherein the 30S subunit undergoes a thermally driven intersubunit rotation and exhibits substantial structural flexibility as well as distinct free energy. We experimentally demonstrate that annealing at a fast cooling rate enhances the 70S ribosome homogeneity and improves local resolution on the 30S subunit. After annealing, the 70S ribosome is in a nonrotated state with respect to corresponding intermediate structures in unannealed or heated ribosomes. Manifold-based analysis further indicates that the annealed 70S ribosome takes a narrow conformational distribution and exhibits a minimum-energy state in the free-energy landscape. Our experimental results offer a facile yet robust approach to enhance protein stability, which is ideal for high-resolution cryogenic electron microscopy. Beyond structure determination, annealing shows great potential for synchronizing proteins on a single-molecule level and can be extended to study protein folding and explore conformational and energy landscapes.

Structural basis for context-specific inhibition of translation by oxazolidinone antibiotics.

The antibiotic linezolid, the first clinically approved member of the oxazolidinone class, inhibits translation of bacterial ribosomes by binding to the peptidyl transferase center. Recent work has demonstrated that linezolid does not inhibit peptide bond formation at all sequences but rather acts in a context-specific manner, namely when alanine occupies the penultimate position of the nascent chain. However, the molecular basis for context-specificity has not been elucidated. Here we show that the second-generation oxazolidinone radezolid also induces stalling with a penultimate alanine, and we determine high-resolution cryo-EM structures of linezolid- and radezolid-stalled ribosome complexes to explain their mechanism of action. These structures reveal that the alanine side chain fits within a small hydrophobic crevice created by oxazolidinone, resulting in improved ribosome binding. Modification of the ribosome by the antibiotic resistance enzyme Cfr disrupts stalling due to repositioning of the modified nucleotide. Together, our findings provide molecular understanding for the context-specificity of oxazolidinones.

Interplay between an ATP-binding cassette F protein and the ribosome from Mycobacterium tuberculosis.

EttA, energy-dependent translational throttle A, is a ribosomal factor that gates ribosome entry into the translation elongation cycle. A detailed understanding of its mechanism of action is limited due to the lack of high-resolution structures along its ATPase cycle. Here we present the cryo-electron microscopy (cryo-EM) structures of EttA from Mycobacterium tuberculosis (Mtb), referred to as MtbEttA, in complex with the Mtb 70S ribosome initiation complex (70SIC) at the pre-hydrolysis (ADPNP) and transition (ADP-VO4) states, and the crystal structure of MtbEttA alone in the post-hydrolysis (ADP) state. We observe that MtbEttA binds the E-site of the Mtb 70SIC, remodeling the P-site tRNA and the ribosomal intersubunit bridge B7a during the ribosomal ratcheting. In return, the rotation of the 30S causes conformational changes in MtbEttA, forcing the two nucleotide-binding sites (NBSs) to alternate to engage each ADPNP in the pre-hydrolysis states, followed by complete engagements of both ADP-VO4 molecules in the ATP-hydrolysis transition states. In the post-hydrolysis state, the conserved ATP-hydrolysis motifs of MtbEttA dissociate from both ADP molecules, leaving two nucleotide-binding domains (NBDs) in an open conformation. These structures reveal a dynamic interplay between MtbEttA and the Mtb ribosome, providing insights into the mechanism of translational regulation by EttA-like proteins.

How to build a ribosome from RNA fragments in Chlamydomonas mitochondria.

Mitochondria are the powerhouse of eukaryotic cells. They possess their own gene expression machineries where highly divergent and specialized ribosomes, named hereafter mitoribosomes, translate the few essential messenger RNAs still encoded by mitochondrial genomes. Here, we present a biochemical and structural characterization of the mitoribosome in the model green alga Chlamydomonas reinhardtii, as well as a functional study of some of its specific components. Single particle cryo-electron microscopy resolves how the Chlamydomonas mitoribosome is assembled from 13 rRNA fragments encoded by separate non-contiguous gene pieces. Additional proteins, mainly OPR, PPR and mTERF helical repeat proteins, are found in Chlamydomonas mitoribosome, revealing the structure of an OPR protein in complex with its RNA binding partner. Targeted amiRNA silencing indicates that these ribosomal proteins are required for mitoribosome integrity. Finally, we use cryo-electron tomography to show that Chlamydomonas mitoribosomes are attached to the inner mitochondrial membrane via two contact points mediated by Chlamydomonas-specific proteins. Our study expands our understanding of mitoribosome diversity and the various strategies these specialized molecular machines adopt for membrane tethering.

Functional role and ribosomal position of the unique N-terminal region of DHX29, a factor required for initiation on structured mammalian mRNAs.

Translation initiation on structured mammalian mRNAs requires DHX29, a DExH protein that comprises a unique 534-aa-long N-terminal region (NTR) and a common catalytic DExH core. DHX29 binds to 40S subunits and possesses 40S-stimulated NTPase activity essential for its function. In the cryo-EM structure of DHX29-bound 43S preinitiation complexes, the main DHX29 density resides around the tip of helix 16 of 18S rRNA, from which it extends through a linker to the subunit interface forming an intersubunit domain next to the eIF1A binding site. Although a DExH core model can be fitted to the main density, the correlation between the remaining density and the NTR is unknown. Here, we present a model of 40S-bound DHX29, supported by directed hydroxyl radical cleavage data, showing that the intersubunit domain comprises a dsRNA-binding domain (dsRBD, aa 377-448) whereas linker corresponds to the long α-helix (aa 460-512) that follows the dsRBD. We also demonstrate that the N-terminal α-helix and the following UBA-like domain form a four-helix bundle (aa 90-166) that constitutes a previously unassigned section of the main density and resides between DHX29's C-terminal α-helix and the linker. In vitro reconstitution experiments revealed the critical and specific roles of these NTR elements for DHX29's function.

New tools for automated cryo-EM single-particle analysis in RELION-4.0.

We describe new tools for the processing of electron cryo-microscopy (cryo-EM) images in the fourth major release of the RELION software. In particular, we introduce VDAM, a variable-metric gradient descent algorithm with adaptive moments estimation, for image refinement; a convolutional neural network for unsupervised selection of 2D classes; and a flexible framework for the design and execution of multiple jobs in pre-defined workflows. In addition, we present a stand-alone utility called MDCatch that links the execution of jobs within this framework with metadata gathering during microscope data acquisition. The new tools are aimed at providing fast and robust procedures for unsupervised cryo-EM structure determination, with potential applications for on-the-fly processing and the development of flexible, high-throughput structure determination pipelines. We illustrate their potential on 12 publicly available cryo-EM data sets.

A cryo-TSEM with temperature cycling capability allows deep sublimation of ice to uncover fine structures in thick cells.

The scanning electron microscope (SEM) has been reassembled into a new type of cryo-electron microscope (cryo-TSEM) by installing a new cryo-transfer holder and anti-contamination trap, which allowed simultaneous acquisition of both transmission images (STEM images) and surface images (SEM images) in the frozen state. The ultimate temperatures of the holder and the trap reached - 190 °C and - 210 °C, respectively, by applying a liquid nitrogen slush. The STEM images at 30 kV were comparable to, or superior to, the images acquired with conventional transmission electron microscope (100 kV TEM) in contrast and sharpness. The unroofing method was used to observe membrane cytoskeletons instead of the frozen section and the FIB methods. Deep sublimation of ice surrounding unroofed cells by regulating temperature enabled to emerge intracellular fine structures in thick frozen cells. Hence, fine structures in the vicinity of the cell membrane such as the cytoskeleton, polyribosome chains and endoplasmic reticulum (ER) became visible. The ER was distributed as a wide, flat structure beneath the cell membrane, forming a large spatial network with tubular ER.

Ribosome Elongation Kinetics of Consecutively Charged Residues Are Coupled to Electrostatic Force.

The speed of protein synthesis can dramatically change when consecutively charged residues are incorporated into an elongating nascent protein by the ribosome. The molecular origins of this class of allosteric coupling remain unknown. We demonstrate, using multiscale simulations, that positively charged residues generate large forces that move the P-site amino acid away from the A-site amino acid. Negatively charged residues generate forces of similar magnitude but move the A- and P-sites closer together. These conformational changes, respectively, increase and decrease the transition state barrier height to peptide bond formation, explaining how charged residues mechanochemically alter translation speed. This mechanochemical mechanism is consistent with in vivo ribosome profiling data exhibiting proportionality between translation speed and the number of charged residues, experimental data characterizing nascent chain conformations, and a previously published cryo-EM structure of a ribosome-nascent chain complex containing consecutive lysines. These results expand the role of mechanochemistry in translation and provide a framework for interpreting experimental results on translation speed.

Structural mechanism of GTPase-powered ribosome-tRNA movement.

GTPases are regulators of cell signaling acting as molecular switches. The translational GTPase EF-G stands out, as it uses GTP hydrolysis to generate force and promote the movement of the ribosome along the mRNA. The key unresolved question is how GTP hydrolysis drives molecular movement. Here, we visualize the GTPase-powered step of ongoing translocation by time-resolved cryo-EM. EF-G in the active GDP-Pi form stabilizes the rotated conformation of ribosomal subunits and induces twisting of the sarcin-ricin loop of the 23 S rRNA. Refolding of the GTPase switch regions upon Pi release initiates a large-scale rigid-body rotation of EF-G pivoting around the sarcin-ricin loop that facilitates back rotation of the ribosomal subunits and forward swiveling of the head domain of the small subunit, ultimately driving tRNA forward movement. The findings demonstrate how a GTPase orchestrates spontaneous thermal fluctuations of a large RNA-protein complex into force-generating molecular movement.