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Selection of the translation start codon is a key step during protein synthesis in human cells. We obtained cryo-EM structures of human 48S initiation complexes and characterized the intermediates of codon recognition by kinetic methods using eIF1A as a reporter. Both approaches capture two distinct ribosome populations formed on an mRNA with a cognate AUG codon in the presence of eIF1, eIF1A, eIF2–GTP–Met-tRNAiMet, and eIF3. The ‘open’ 40S subunit conformation differs from the human 48S scanning complex and represents an intermediate preceding the codon recognition step. The ‘closed’ form is similar to reported structures of complexes from yeast and mammals formed upon codon recognition, except for the orientation of eIF1A, which is unique in our structure. Kinetic experiments show how various initiation factors mediate the population distribution of open and closed conformations until 60S subunit docking. Our results provide insights into the timing and structure of human translation initiation intermediates and suggest the differences in the mechanisms of start codon selection between mammals and yeast.

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.

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.

SummarySingle particle cryo-EM is a powerful method to solve the three-dimensional structures of biological macromolecules. The technological development of electron microscopes, detectors, automated procedures in combination with user friendly image processing software and ever-increasing computational power have made cryo-EM a successful and largely expanding technology over the last decade. At resolutions better than 4 Å, atomic model building starts becoming possible but the direct visualization of true atomic positions in protein structure determination requires significantly higher (< 1.5 Å) resolution, which so far could not be attained by cryo-EM. The direct visualization of atom positions is essential for understanding protein-catalyzed chemical reaction mechanisms and to study drug-binding and -interference with protein function. Here we report a 1.25 Å resolution structure of apoferritin obtained by cryo-EM with a newly developed electron microscope providing unprecedented structural details. Our apoferritin structure has almost twice the 3D information content of the current world record reconstruction (at 1.54 Å resolution1). For the first time in cryo-EM we can visualize individual atoms in a protein, see density for hydrogen atoms and single atom chemical modifications. Beyond the nominal improvement in resolution we can also show a significant improvement in quality of the cryo-EM density map which is highly relevant for using cryo-EM in structure-based drug design.

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 decade1. At resolutions better than 4 A, 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 A) 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 proteins2. Here we report a 1.25 A-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 A resolution3). 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.

SummaryWe developed a method, named GraDeR, which substantially improves the preparation of membrane protein complexes for structure determination by single-particle cryo-electron microscopy (cryo-EM). In GraDeR, glycerol gradient centrifugation is used for the mild removal of free detergent monomers and micelles from lauryl maltose-neopentyl glycol detergent stabilized membrane complexes, resulting in monodisperse and stable complexes to which standard processes for water-soluble complexes can be applied. We demonstrate the applicability of the method on three different membrane complexes, including the mammalian FoF1 ATP synthase. For this highly dynamic and fragile rotary motor, we show that GraDeR allows visualizing the asymmetry of the F1 domain, which matches the ground state structure of the isolated domain. Therefore, the present cryo-EM structure of FoF1 ATP synthase provides direct structural evidence for Boyer's binding change mechanism in the context of the intact enzyme.

Mitochondrial FoF1ATP synthase is a membrane bound molecular machine central to cellular energy conversion and cristae architecture. Recently, a novel domain has been visualized in the intermembrane space region of mammalian ATP synthase. The complete three-dimensional (3D) structure, composition and function of this domain - which we term intermembrane space domain (IMD) - are unknown. Here, we present two distinct 3D structures of monomeric bovine FoF1ATP synthase by single particle cryo-electron microscopy (cryo-EM) that differ by the presence and absence of the IMD. Comparison of both structures reveals the IMD to be a bipartite and weakly associated domain of FoF1ATP synthase. The tubular sub-domain of the IMD appears to contact the rotor-ring region, its globular sub-domain is anchored in the membrane-bending kink of the ATP synthase. However, absence of the IMD does not impact the kink in the transmembrane region ruling out a functional role in membrane bending. By combining our structural analysis with chemical cross-linking and reported biochemical, genetic and structural data we identify 6.8PL and DAPIT as the subunits forming the intermembrane space domain. We compare the present structure of the mammalian IMD in the bovine FoF1ATP synthase monomer with structures of dimeric FoF1ATP synthase from yeast and ciliate showing that the IMD is a common, but structurally divergent feature of several mitochondrial ATP synthases. On the basis of our analysis we discuss potential functions of the novel domain in rotary catalysis, oligomerization and mitochondrial permeability transition.

SelB is an elongation factor specialized to deliver the selenocysteine (Sec) tRNA to the ribosome by recoding the UGA stop codon on the mRNA. Initially the tRNA is in complex with selB and GTP forming the ternary complex (TC). High-resolution cryo-EM structures of intermediates of the Sec incorporation pathway uncover large-scale conformational changes of the ribosome and the TC. To complement the structural information with energetics and rapid dynamics, we performed extensive all-atom molecular dynamics simulations of the ribosome with bound TC as well as of the free TC in solution. The simulations of the free TC were started after extracting the TC from the ribosome-bound cryo-EM structures. The TC was found to rapidly interconvert between the different conformations allowing us to construct the free-energy landscape of the involved motions. This free-energy landscape indicates that the intrinsic large-scale conformational changes of the tRNA and SelB during the delivery to the ribosome are not rate-limiting to the process. In simulations of the free TC started from the GTPase-activated ribosome-bound conformation, the TC rapidly transitions into an inactivated conformation, showing that the GTPase-activated state is strongly stabilized by the ribosome. The simulations of the full ribosome with bound TC in the intermediate states allow us to identify the motions that are rate-limiting to the process of tRNA delivery and to identify the molecular mechanism of the domain closure of small ribosomal subunit upon tRNA decoding.

During protein synthesis, tRNAs move from the ribosome's aminoacyl to peptidyl to exit sites. Here we investigate conformational motions during spontaneous translocation, using molecular dynamics simulations of 13 intermediate-translocation-state models obtained by combining Escherichia coli ribosome crystal structures with cryo-EM data. Resolving fast transitions between states, we find that tRNA motions govern the transition rates within the pre- and post-translocation states. Intersubunit rotations and L1-stalk motion exhibit fast intrinsic submicrosecond dynamics. The L1 stalk drives the tRNA from the peptidyl site and links intersubunit rotation to translocation. Displacement of tRNAs is controlled by 'sliding' and 'stepping' mechanisms involving conserved L16, L5 and L1 residues, thus ensuring binding to the ribosome despite large-scale tRNA movement. Our results complement structural data with a time axis, intrinsic transition rates and molecular forces, revealing correlated functional motions inaccessible by other means.

In all domains of life, selenocysteine (Sec) is delivered to the ribosome by selenocysteine-specific tRNA (tRNASec) with the help of a specialized translation factor, SelB in bacteria. Sec-tRNASec 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-tRNASec binding by SelB and show large-scale rearrangements of Sec-tRNASec. Upon initial binding of SelB-Sec-tRNASec to the ribosome and codon reading, the 30S subunit adopts an open conformation with Sec-tRNASec 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-tRNASec 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.

During the translocation step of protein synthesis, a complex of two transfer RNAs bound to messenger RNA (tRNA-mRNA) moves through the ribosome. The reaction is promoted by an elongation factor, called EF-G in bacteria, which, powered by GTP hydrolysis, induces an open, unlocked conformation of the ribosome that allows for spontaneous tRNA-mRNA movement. Here we show that, in the absence of EF-G, there is spontaneous backward movement, or retrotranslocation, of two tRNAs bound to mRNA. Retrotranslocation is driven by the gain in affinity when a cognate E-site tRNA moves into the P site, which compensates the affinity loss accompanying the movement of peptidyl-tRNA from the P to the A site. These results lend support to the diffusion model of tRNA movement during translocation. In the cell, tRNA movement is biased in the forward direction by EF-G, which acts as a Brownian ratchet and prevents backward movement.

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.

Molecular machines or macromolecular complexes are supramolecular assemblies of biomolecules that ensure cellular homeostasis. Structure determination of those complexes in a purified state is often a tedious undertaking due to the compositional complexity and the related relative structural instability. To improve the stability of macromolecular complexes in vitro, we present here a generic method that optimizes the stability, homogeneity and solubility of macromolecular complexes by sparse-matrix screening of their thermal unfolding behaviour in the presence of various buffers and small molecules. The method includes the automated analysis of thermal unfolding curves based on a newly developed biophysical unfolding model for complexes. We found that under stabilizing conditions even large multi-component complexes reveal an almost ideal two-state unfolding behaviour. We envisage an improved biochemical understanding of purified macromolecules as well as a substantial boost in successful macromolecular complex structure determination by both X-ray crystallography and Cryo EM.

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-tRNA Sec 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’.

A single particle cryo-EM structure of the 70S ribosome in complex with the elongation factor Tu breaks the 3 A 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. 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 resolution1,2 and all structures available to date have been limited to resolutions above 3 A3,4. 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 A resolution using spherical aberration (Cs)-corrected cryo-EM. Overall, the cryo-EM reconstruction at 2.9 A resolution is comparable to the best-resolved X-ray structure of the E. coli 70S ribosome5 (2.8 A), but provides more detailed information (2.65 A) 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.

After peptide bond formation the transfer RNAs (tRNAs) bound to the ribosome translocate by more than 7 nm to adjacent binding sites, accompanied by large-scale conformational motions of the ribosome. Combining cryo-EM reconstructions of translocation intermediates (Fischer, Nature 2010) with high resolution crystal structures, we obtained 13 near-atomic resolution structures. The quality of these structures was validated using recent crystal structures and subsequently all-atom molecular dynamics simulations of the fully solvated 70S ribosome were carried out for each of the 13 intermediate states, totaling 1.5 µs. The obtained dynamics within the intermediate states allowed us to estimate transition rates between states for motions of the L1-stalk, tRNAs and intersubunit rotations. These rates revealed rapid motions of the L1-stalk and the small subunit on sub-microsecond timescales, whereas the tRNA motions were seen to be rate-limiting for most transitions. By calculating the free energy of interaction between L1-stalk and tRNA, we obtained molecular forces revealing that the L1-stalk is actively pulling the tRNA from P to E site, thereby overcoming barriers for the tRNA motion. Further, ribosomal proteins L5 and L16 guide the tRNAs by ‘sliding’ and ‘stepping’ mechanisms involving key protein-tRNA contacts, explaining how tRNA binding affinity is kept sufficiently constant to allow rapid translocation despite large-scale displacements.

Among the translation factors that assist the ribosome in synthesizing proteins, elongation factor G (EF-G) is the only one that functions in two different phases of protein synthesis, i. e. in the translocation step of the elongation phase and in ribosome disassembly following termination. During translocation two tRNAs move by large distances from one site of the ribosome to the next, adjacent site, with the coupled movement of mRNA by one codon. The process is promoted by EF-G and GTP hydrolysis to proceed at the velocity required for rapid protein synthesis in the cell. During ribosome recycling the ribosomal post-termination complex is dissociated into subunits; the reaction is brought about by EF-G together with the ribosome recycling factor (RRF) and requires GTP hydrolysis. Fundamental questions in understanding EF-G function are: (i) How does EF-G accelerate the movement of tRNAs together with the mRNA on the ribosome; (ii) how does EF-G cooperate with RRF to dissociate the ribosomes; and (iii) how are GTP hydrolysis and Pi release coupled to forward movement and ribosome disassembly? The aim of this review is to summarize the recent insights into the molecular mechanism of translocation and ribosome recycling and the role of EF-G in the two reactions. Detailed accounts focusing on different aspects of translocation can also be found in several recent reviews (Shoji et al., 2009; Dunkle and Cate, 2010; Frank and Gonzalez, 2010).

We developed a method, named GraFix, that considerably improves sample quality for structure determination by single-particle electron cryomicroscopy (cryo-EM). GraFix uses a glycerol gradient centrifugation step in which the complexes are centrifuged into an increasing concentration of a chemical fixation reagent to prevent aggregation and to stabilize individual macromolecules. The method can be used to prepare samples for negative-stain, cryo-negative-stain and, particularly, unstained cryo-EM.

In bacteria, UGA stop codons can be recoded to direct the incorporation of selenocysteine into proteins on the ribosome. Recoding requires a selenocysteine incorporation sequence (SECIS) downstream of the UGA codon, a specialized translation factor SelB, and the non-canonical Sec-tRNASec, which is formed from Ser-tRNASec by selenocysteine synthase, SelA, using selenophosphate as selenium donor. Here we describe a rapid-kinetics approach to study the mechanism of selenocysteine insertion into proteins on the ribosome. Labeling of SelB, Sec-tRNASec and other components of the translational machinery allows direct observation of the formation or dissociation of complexes by monitoring changes in the fluorescence of single dyes or fluorescence resonance energy transfer between two fluorophores. Furthermore, the structure of SelA was studied by electron cryomicroscopy (cryo-EM). We report that intact SelA from the thermophilic bacterium Moorella thermoacetica (mthSelA) can be vitrified for cryo-EM using a controlled-environment vitrification system. Two-dimensional image analysis of vitrified mthSelA images shows that SelA can adopt the wide range of orientations required for high-resolution structure determination by cryo-EM. The results indicate that mthSelA forms a homodecamer that has a ring-like structure with five bilobed wings, similar to the structure of the E. coli complex determined previously.

Summary The L7/12 stalk of the large subunit of bacterial ribosomes encompasses protein L10 and multiple copies of L7/12. We present crystal structures of Thermotoga maritima L10 in complex with three L7/12 N-terminal-domain dimers, refine the structure of an archaeal L10E N-terminal domain on the 50S subunit, and identify these elements in cryo-electron-microscopic reconstructions of Escherichia coli ribosomes. The mobile C-terminal helix α8 of L10 carries three L7/12 dimers in T. maritima and two in E. coli, in concordance with the different length of helix α8 of L10 in these organisms. The stalk is organized into three elements (stalk base, L10 helix α8-L7/12 N-terminal-domain complex, and L7/12 C-terminal domains) linked by flexible connections. Highly mobile L7/12 C-terminal domains promote recruitment of translation factors to the ribosome and stimulate GTP hydrolysis by the ribosome bound factors through stabilization of their active GTPase conformation.

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.

During the elongation cycle, after peptide-bonds are formed in the ribosome, transfer RNAs translocate to their new binding sites. Resting on extensive MD simulations of 13 near-atomistically resolved translocation intermediates of the fully solvated ribosome, we have estimated the rates from transition state theory for the motions of the tRNAs, 30S head and body, as well as the L1-stalk. The Kramers pre-factor and transmission coefficient were determined from a statistical analysis of transitions observed in the simulations.To that aim, we first estimated all free energy barrier heights from a multidimensional quasi-harmonic approximation derived from local fluctuation analysis. Second, we introduced two model parameters, an attempt rate and a constant scaling factor for the estimated barrier heights. using the assumption that all barrier crossings occur at the same attempt rate, scaling factor and attempt rate were obtained through a least squares fitting of the transition probabilities observed in our simulation times to the respective transition probabilities from Kramers theory.The obtained rate estimates range from ns to ms and suggest that tRNA movement, rather than body and head rotation, is rate-limiting for most transitions between intermediate states of tRNA translocation.

During protein synthesis, the ribosome undergoes large-scale conformational changes. In particular, the process of tRNA translocation is accompanied by concerted rotations of the 30S ribosomal subunit of more than 20 degrees relative to the 50S. At each step of the translocation process, affinity between the two independent subunits must be finely tuned in order to allow such high conformational flexibility while still maintaining integrity of the ribosomal complex. How the ribosome achieves this is still not fully understood. We address this question by extensive all-atom, explicit solvent molecular dynamics simulations (total simulation time > 1.5 μs) of the 70S ribosome starting from 13 distinct translocation intermediates. The resulting structural ensemble provides a detailed picture of translocation dynamics. Analysis of the trajectories at the residue level reveals two classes of intersubunit contact interactions: i) persistent residue contacts which are independent of 30S rotation and located close to the axis of rotation. ii) state specific contacts, seen mostly on the periphery. The baseline of the interaction energy needed to maintain the ribosomal assembly is provided by class i) interactions, while the rupture and formation of class ii) contacts contributes low changes to the overall interaction energy and may serve as an affinity tuning mechanism in switching between different translocation states. Our simulations reveal decreased amplitude of the intersubunit rotations and weakening of stabilizing peripheral contacts upon removal of the tRNAs. This finding is confirmed by cryo-EM analysis of tRNA depleted ribosomes.

In the process of ribosomal translocation, two tRNAs must move through the ribosome, adopting a number of different conformations. To obtain atomistic descriptions of this highly dynamic process, we fitted high-resolution X-ray structures to a set of 23 cryo-EM density maps of the ribosome [1]. From a subset of these we started all-atom molecular dynamics simulations of the solvated 70S ribosome as well as of the two tRNAs in solvent. The simulations provided estimates of the conformational free energy needed to bring the tRNAs from solvent into the ribosome at different stages of the translocation pathway. Our results suggest that the tRNAs store conformational free energy during translocation, which is used to drive translocation of tRNAs and released upon exiting the ribosome. Additionally, as the tRNAs translocate, they form a complex interaction network with a number of ribosomal proteins and rRNA helices. Our simulations also enabled us to estimate contact energies for these interactions, which offer a detailed picture of tRNA handovers, e.g. by the ribosomal proteins L16 and L5 and the L1-stalk. Two of our structures (pre1 and pre4 states) agree with the subsequently determined X-ray structures [2] to within 5A supporting the validity of our approach.[1] Fisher et al, Nature 2010.[2] Dunkle et al, Science 2011.

Biomachines such as the ribosome undergo substantial conformational changes during their work cycle. During ribosomal translocation, in particular, a broad variety of functional contact patches dynamically form and rupture, the detailed characterization of which is still lacking. Here we use extended atomistic simulations of the whole solvated ribosome, starting from 13 distinct translocational substates, to obtain a comprehensive picture of these contact patches. To that aim, we developed a novel analysis tool, which is broadly applicable to large flexible parts of simulated biomolecules, and enabled us to quantitatively extract contact occupancies and changes for all available conformational states.For the 13 translocational sub-states of the ribosome, molecular dynamics simulations yielded extended all-atom trajectories. From these trajectories the frequency of all possible inter-atomic contacts between the 30S and 50S subunits was determined. As the search for atom contacts scales with the number of particles and simulation length, a fast, hierarchical algorithm based on kd-tree branch exclusion was developed and applied. Subsequently, contacting atom pairs were filtered according to contact frequency and then assigned to residues. From this information a graph was constructed whose edges connect contacting residues. The regions identified form this analysis provided a rigorous, intuitive, and comprehensive picture of ribosomal contact patches during transloaction, and explained how the ribosome maintains its fine-tuned intersubunit affinity despite drastic conformational changes.

Translocation of tRNAs through the ribosome is accompanied by large-scale highly concerted conformational motions. We obtained 23 near-atomic resolution structures of translocation conformational substates by combining cryo-EM densities (Fischer, Nature, 2010) with high-resolution X-ray structures. For 13 structures, we carried out extensive molecular dynamics simulations of the fully solvated 70S ribosome, totaling 1.5μs. The obtained structural ensemble, together with the 23 static structures, offers a most complete all-atom picture of ribosomal translocation dynamics. The simulations captured sufficient conformational dynamics to estimate free energy barriers between states, suggesting a hierarchy of timescales for motions of the L1-stalk, tRNAs, and intersubunit rotations. Interaction energies derived from the simulations allowed us to characterize molecular driving forces; e.g., the L1-stalk actively pulls the tRNA from the P- to the E-site, rather than being pushed or passively co-translocated. Addressing the question of how the affinity between the two ribosomal subunits is fine-tuned despite rotations of more than 20 degrees, intersubunit contacts were found to fall into two classes: i) contacts which persist throughout the different states, independent of intersubunit rotations. ii) contacts that are specific to certain states. Persisting contacts are seen close to the axis of rotation and contribute to the baseline of intersubunit interaction energy. In contrast, contacts of residues situated on the periphery are found to be mostly state-specific. The rupture and formation of state-specific contacts entails low changes of the overall interaction energy, allowing the subunits to remain assembled. Key contacts in the periphery predict and explain the decreased amplitude of the intersubunit rotations seen by cryo-EM in the absence of tRNAs. These results suggest further mutations that should stabilize or destabilize specific intermediate states of ribosomal translocation.

During the elongation cycle, after peptide-bonds are formed in the ribosome, transfer RNAs translocate to their new binding sites. Combining high-resolution crystal structures, cryo-EM data of multiple translocation intermediates of factorless retro-translocation, and atomistic molecular dynamics simulations, we have analysed collective motions, intrinsic time scales, and overall transition rates of ribosomal motions promoting tRNA translocation.From a Marcus-Theory type transition state analysis of fast molecular fluctuations, unexpectedly fast sub-microsecond intrinsic rates were resolved for body and head rotations as well as swiveling motions. The tRNA transitions between A,P, and E site are seen to be clearly slower than microseconds and seem to determine the overall transition rate between the intermediates. Remarkably, our kinetic analysis recovers the sequence of intermediates proposed in Fischer et al. that was purely based on structural similarity, thereby adding time information to these data.Together with the millisecond dynamics revealed from single molecule studies and the slow dynamics between pre and post states, a Frauenfelder-type hierarchy of time scales and corresponding free energy barriers emerges, underscoring the notion of the ribosome as a stochastic molecular machine.

We combined high-resolution crystal structures with cryo-EM maps of 13 intermediate states of ribosomal factorless spontaneous retro-translocation to obtain structures of the 70S ribosome in each of the 13 states in atomic detail. We then performed 100 ns all-atom, explicit-solvent molecular dynamics simulations for each of these states. Intrinsic rates for key ribosomal motions between the states were estimated from the short time fluctuations of the L1-stalk, the tRNAs and intersubunit rotations. The rates revealed rapid, sub-microsecond motions of the L1-stalk and the 30S subunit. Surprisingly, it is tRNA motions, rather than large-scale intersubunit rotations, that are rate limiting for most transitions. The interaction free energy profile of the L1-stalk with the tRNA obtained from additional umbrella sampling simulations of the L1-stalk/tRNA interactions revealed an active role of the L1-stalk in pulling the tRNA from the P to the E site. Further, by detailed analysis of the frequency of contacts between 50S ribosomal proteins L5 and L16 and the tRNAs, we identified specific residues which guide the tRNAs between the binding sites. A sequence analysis of the L1, L5 and L16 proteins revealed that the conservation score for contacting residues is significantly above average. Different types of contacts characterize the interplay of these proteins with the tRNAs and involve 1) sliding of L5 residues along the tRNA elbow 2) stepping of the tRNA between L16 contact patches 3) final pulling of the tRNA by the L1-stalk. These contact mechanisms can explain how both rapid translocation and a stable tRNA binding affinity can be achieved despite large-scale displacements.