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Single-particle electron cryo-microscopy (cryo-EM) is a powerful method for solving the three-dimensional structures of biological macromolecules. The technological development of transmission electron microscopes, detectors and automated procedures in combination with user-friendly image processing software and ever-increasing computational power have made cryo-EM a successful and expanding technology over the past decade 1 . At resolutions better than 4 Å, atomic model building starts to become possible, but the direct visualization of true atomic positions in protein structure determination requires much higher (better than 1.5 Å) resolution, which so far has not been attained by cryo-EM. The direct visualization of atom positions is essential for understanding the mechanisms of protein-catalysed chemical reactions, and for studying how drugs bind to and interfere with the function of proteins 2 . Here we report a 1.25 Å-resolution structure of apoferritin obtained by cryo-EM with a newly developed electron microscope that provides, to our knowledge, unprecedented structural detail. Our apoferritin structure has almost twice the 3D information content of the current world record reconstruction (at 1.54 Å resolution 3 ). We can visualize individual atoms in a protein, see density for hydrogen atoms and image single-atom chemical modifications. Beyond the nominal improvement in resolution, we also achieve a substantial improvement in the quality of the cryo-EM density map, which is highly relevant for using cryo-EM in structure-based drug design. Advances in electron cryo-microscopy allow the structure of apoferritin to be determined at a resolution that enables the visualization of individual atoms.

A single particle cryo-EM structure of the 70S ribosome in complex with the elongation factor Tu breaks the 3 Å resolution barrier of the technique and locally exceeds the resolution of previous crystallographic studies, revealing all modifications in rRNA and explaining their roles in ribosome function and antibiotic binding. Ribosome–EF-Tu complex structure One of the cell's largest and most important macromolecular complexes, the ribosome has been the target of intensive structural study. Until now, crystallographic studies have provided the highest resolution images of this complex. Now Holger Stark and colleagues have used the latest single-particle electron cryomicroscopy approaches to characterize the Escherichia coli 70S ribosome bound to the Tu elongation factor, a charged tRNA, and the antibiotic kirromycin, at a resolution that locally exceeds that obtained crystallographically. Novel insights are obtained about the modifications occurring on the rRNA and about the more flexible regions of the protein that are inaccessible to crystallographic analysis. Single particle electron cryomicroscopy (cryo-EM) has recently made significant progress in high-resolution structure determination of macromolecular complexes due to improvements in electron microscopic instrumentation and computational image analysis. However, cryo-EM structures can be highly non-uniform in local resolution 1 , 2 and all structures available to date have been limited to resolutions above 3 Å 3 , 4 . Here we present the cryo-EM structure of the 70S ribosome from Escherichia coli in complex with elongation factor Tu, aminoacyl-tRNA and the antibiotic kirromycin at 2.65–2.9 Å resolution using spherical aberration (C s )-corrected cryo-EM. Overall, the cryo-EM reconstruction at 2.9 Å resolution is comparable to the best-resolved X-ray structure of the E. coli 70S ribosome 5 (2.8 Å), but provides more detailed information (2.65 Å) at the functionally important ribosomal core. The cryo-EM map elucidates for the first time the structure of all 35 rRNA modifications in the bacterial ribosome, explaining their roles in fine-tuning ribosome structure and function and modulating the action of antibiotics. We also obtained atomic models for flexible parts of the ribosome such as ribosomal proteins L9 and L31. The refined cryo-EM-based model presents the currently most complete high-resolution structure of the E. coli ribosome, which demonstrates the power of cryo-EM in structure determination of large and dynamic macromolecular complexes.

Elongation factors Tu (EF-Tu) and SelB are translational GTPases that deliver aminoacyl-tRNAs (aa-tRNAs) to the ribosome. In each canonical round of translation elongation, aa-tRNAs, assisted by EF-Tu, decode mRNA codons and insert the respective amino acid into the growing peptide chain. Stop codons usually lead to translation termination; however, in special cases UGA codons are recoded to selenocysteine (Sec) with the help of SelB. Recruitment of EF-Tu and SelB together with their respective aa-tRNAs to the ribosome is a multistep process. In this review, we summarize recent progress in understanding the role of ribosome dynamics in aa-tRNA selection. We describe the path to correct codon recognition by canonical elongator aa-tRNA and Sec-tRNASec and discuss the local and global rearrangements of the ribosome in response to correct and incorrect aa-tRNAs. We present the mechanisms of GTPase activation and GTP hydrolysis of EF-Tu and SelB and summarize what is known about the accommodation of aa-tRNA on the ribosome after its release from the elongation factor. We show how ribosome dynamics ensures high selectivity for the cognate aa-tRNA and suggest that conformational fluctuations, induced fit and kinetic discrimination play major roles in maintaining the speed and fidelity of translation. This article is part of the themed issue ‘Perspectives on the ribosome’.

The translocation step of protein synthesis entails large-scale rearrangements of the ribosome–transfer RNA (tRNA) complex. Here we have followed tRNA movement through the ribosome during translocation by time-resolved single-particle electron cryomicroscopy (cryo-EM). Unbiased computational sorting of cryo-EM images yielded 50 distinct three-dimensional reconstructions, showing the tRNAs in classical, hybrid and various novel intermediate states that provide trajectories and kinetic information about tRNA movement through the ribosome. The structures indicate how tRNA movement is coupled with global and local conformational changes of the ribosome, in particular of the head and body of the small ribosomal subunit, and show that dynamic interactions between tRNAs and ribosomal residues confine the path of the tRNAs through the ribosome. The temperature dependence of ribosome dynamics reveals a surprisingly flat energy landscape of conformational variations at physiological temperature. The ribosome functions as a Brownian machine that couples spontaneous conformational changes driven by thermal energy to directed movement. The ribosome in motion During protein synthesis, transfer RNAs move sequentially through the A, P and E sites of the ribosome as their attached amino acids are transferred to the growing peptide chain. Large conformational movements accompany their translocation. Holger Stark and colleagues have processed a staggering 1.9 million single-particle electron cryomicroscopy images of the ribosome to visualize these dynamic changes. They conclude that the conformational changes are thermally driven (or 'Brownian') and that they cause directed movement of transfer RNAs on a narrow path through the ribosome. During protein synthesis within the ribosome, transfer RNAs (tRNAs) move sequentially through different sites as their attached amino acids are transferred onto the growing protein chain. Large conformational movements accompany this process. Here, a staggering 1.9 million electron cryomicroscopy images of the ribosome have been processed to visualize these changes. The results reveal that the ribosome functions as a Brownian machine that couples spontaneous changes driven by thermal energy to directed 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. Movement of the ribosome along an mRNA requires the universally-conserved translocase (EF-G in bacteria) that couples GTP hydrolysis to directed movement. Here the authors use time-resolved Cryo-EM to visualize the GTPase-powered step on native translocating ribosomes and capture key translocation intermediates.

In all domains of life, selenocysteine (Sec) is delivered to the ribosome by selenocysteine-specific tRNA (tRNA Sec ) with the help of a specialized translation factor, SelB in bacteria. Sec-tRNA Sec recodes a UGA stop codon next to a downstream mRNA stem–loop. Here we present the structures of six intermediates on the pathway of UGA recoding in Escherichia coli by single-particle cryo-electron microscopy. The structures explain the specificity of Sec-tRNA Sec binding by SelB and show large-scale rearrangements of Sec-tRNA Sec . Upon initial binding of SelB–Sec-tRNA Sec to the ribosome and codon reading, the 30S subunit adopts an open conformation with Sec-tRNA Sec covering the sarcin–ricin loop (SRL) on the 50S subunit. Subsequent codon recognition results in a local closure of the decoding site, which moves Sec-tRNA Sec away from the SRL and triggers a global closure of the 30S subunit shoulder domain. As a consequence, SelB docks on the SRL, activating the GTPase of SelB. These results reveal how codon recognition triggers GTPase activation in translational GTPases. The structures of several states on the pathway of SelB-mediated delivery of selenocysteine-specific tRNA to the ribosome in Escherichia coli reveal the mechanism of UGA stop codon recoding to selenocysteine and show how codon recognition triggers activation of translational GTPases. GTPase activation of elongation factor SelB In some messenger RNAs, the UGA stop codon is recoded using the specialized amino acid selenocysteine (Sec), bound to Sec-specific tRNA (tRNA Sec ). The recoding process also requires the SelB GTPase. Holger Stark and colleagues have solved multiple structures of the Escherichia coli ribosome bound to Sec-tRNA Sec and SelB to understand how SelB interacts with the tRNA, and how this complex reorders both the small and large subunits of the ribosome upon binding. The way in which these events at the codon activate the GTPase is revealed.

Alternative ribosome-rescue factor B (ArfB) rescues ribosomes stalled on non-stop mRNAs by releasing the nascent polypeptide from the peptidyl-tRNA. By rapid kinetics we show that ArfB selects ribosomes stalled on short truncated mRNAs, rather than on longer mRNAs mimicking pausing on rare codon clusters. In combination with cryo-electron microscopy we dissect the multistep rescue pathway of ArfB, which first binds to ribosomes very rapidly regardless of the mRNA length. The selectivity for shorter mRNAs arises from the subsequent slow engagement step, as it requires longer mRNA to shift to enable ArfB binding. Engagement results in specific interactions of the ArfB C-terminal domain with the mRNA entry channel, which activates peptidyl-tRNA hydrolysis by the N-terminal domain. These data reveal how protein dynamics translate into specificity of substrate recognition and provide insights into the action of a putative rescue factor in mitochondria. Rescue of ribosomes stalled on non-stop mRNA is essential for cell viability, and several rescue systems to resolve stalling exist in bacteria. Here, the authors use rapid kinetics and cryo-EM to reveal the pathway and selectivity mechanism of ArfB-mediated ribosome rescue.

Antibiotic resistance is a growing threat, underscoring the need to understand the underlying mechanisms. Aminoglycosides kill bacteria by disrupting translation fidelity, leading to the synthesis of aberrant proteins. Surprisingly, mutations in fusA , a gene encoding translation elongation factor G (EF-G), frequently confer resistance, even though EF-G neither participates in mRNA decoding nor blocks aminoglycoside binding. Here, we show that EF-G resistance variants selectively slow ribosome movement along mRNA when aminoglycosides are bound. This delay increases the chance that the drug dissociates before misreading occurs. Over several elongation cycles, this selective silencing of drug-bound ribosomes prevents error cluster formation, preserving proteome and membrane integrity. As a result, fusA mutations confer resistance early in treatment by preventing self-promoted aminoglycoside uptake. Translation on drug-free ribosomes remains sufficiently rapid to sustain near-normal bacterial growth. The mechanism of selective silencing of corrupted targets reveals a previously unrecognized antibiotic resistance strategy with potential therapeutic implications. Mutations in elongation factor G protect bacteria from aminoglycoside antibiotics through unknown mechanisms. Here, the authors show that the mutations selectively slow the movement of antibiotic-bound ribosomes along mRNA, which prevents error-prone protein synthesis and thus membrane damage and antibiotic uptake.

The coupled translocation of transfer RNA and messenger RNA through the ribosome entails large-scale structural rearrangements, including step-wise movements of the tRNAs. Recent structural work has visualized intermediates of translocation induced by elongation factor G (EF-G) with tRNAs trapped in chimeric states with respect to 30S and 50S ribosomal subunits. The functional role of the chimeric states is not known. Here we follow the formation of translocation intermediates by single-molecule fluorescence resonance energy transfer. Using EF-G mutants, a non-hydrolysable GTP analogue, and fusidic acid, we interfere with either translocation or EF-G release from the ribosome and identify several rapidly interconverting chimeric tRNA states on the reaction pathway. EF-G engagement prevents backward transitions early in translocation and increases the fraction of ribosomes that rapidly fluctuate between hybrid, chimeric and posttranslocation states. Thus, the engagement of EF-G alters the energetics of translocation towards a flat energy landscape, thereby promoting forward tRNA movement. EF-G enhances the rate of tRNA–mRNA translocation on the ribosome. Here the authors use single-molecule FRET to follow tRNA translocation in real time, identifying new chimeric intermediates and suggesting how EF-G binding and GTP hydrolysis change the energetic landscape of translocation to accelerate forward tRNA movement.

Theory predicts the existence of barrierless protein folding. Without barriers, folding should be noncooperative and the degree of native structure should be coupled to overall protein stability. We investigated the thermal unfolding of the peripheral subunit binding domain from Escherichia coli's 2-oxoglutarate dehydrogenase multienzyme complex (termed BBL) with a combination of spectroscopic techniques and calorimetry. Each technique probed a different feature of protein structure. BBL has a defined three-dimensional structure at low temperatures. However, each technique showed a distinct unfolding transition. Global analysis with a statistical mechanical model identified BBL as a downhill-folding protein. Because of BBL's biological function, we propose that downhill folders may be molecular rheostats, in which effects could be modulated by altering the distribution of an ensemble of structures.