Spontaneous Intersubunit Rotation in Single Ribosomes

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A macromolecular X-ray crystal structure is usually represented as a single static model with a single set of temperature factors representing a simple approximation of motion and disorder of the structure. Multiconformer representations of small proteins have been shown to better describe anisotropic motion and disorder and improve the quality of their electron density maps. Here, we apply multistart simulated annealing crystallographic refinement to a 70S ribosome-RF1 translation termination complex that was recently solved at 3.2 Å resolution. The analysis improves the interpretability of the electron density map of this 2.5-MDa ribonucleoprotein complex and provides insights into its structural dynamics. We also used multistart refinement and conventional Fourier difference maps to address a recent study in which cross-crystal averaging between two crystal forms of the 70S ribosome was used to evaluate reported differences between two ribosome crystal structures solved at 2.8 and 3.7 Å resolution. Our analysis suggests that results obtained from cross-crystal averaging are inherently biased toward the higherresolution dataset.multistart refinement ͉ protein synthesis ͉ ribosome dynamics ͉ rRNA C rystallographic techniques and methods of structure determination have progressed rapidly in recent years, accompanied by an explosion in the amount of structural information about biological molecules and macromolecular assemblies. X-ray crystal structures have traditionally been represented by a single set of atomic coordinates accompanied by a set of temperature factors (B factors) to characterize atomic thermal motion. At most experimentally attainable resolutions, a spherical approximation for thermal motion is used, accounting only for the isotropic component of atomic displacement. It has been shown that the use of a static model with isotropic thermal parameters underestimates the total disorder and conformational variability of macromolecules (1-5). The limitation of the single-model approach is exemplified by several cases where high-resolution data are available. Nearly half the amino acid side chains and even some backbone atoms were found to have alternative conformations in the 0.54 Å structure of crambin and in the 1.0 Å structure of calmodulin (1, 6). To account for anharmonic contributions and conformational variability in X-ray structures, the use of an ensemble of models, as has long been standard practice in structure determination by NMR, has been suggested as a replacement for the traditional single-model approach (7). Multiconformer refinement has been implemented using both real-and reciprocal-space refinement approaches (2,3,(8)(9)(10)(11)(12)(13) and was demonstrated to yield improved agreement with diffraction data in most cases. In the case of molecular replacement phasing, electron density maps are often biased toward the search model; when a structure ensemble is used, however, model bias can be reduced significantly (14).In X-ray structures of the ribosome, the largest asymmetric struct...

Section: Resultssupporting

confidence: 62%

Section: Evaluating Differences Between 28 å and 37 å Structures Ofmentioning

confidence: 99%

Multistart simulated annealing refinement of the crystal structure of the 70S ribosome

Коростелев

Laurberg

Noller

2009

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“…Because the ribosomes captured in our snapshots were not engaged in protein synthesis, lacking mRNA and aminoacylated tRNAs, we must assume that their idle motions in the thermal environment sample the conformational space permitted by the degrees of freedom, thus exhibiting the conformational changes that would be productive in the presence of the ligands of the translational machinery (mRNA, aa-tRNA, eEF2, and eEF1A). Indeed, idling of the pretranslocational ribosome in the absence of elongation factors along the direction of the most prominent conformational changes (intersubunit motion and opening/ closing of L1 stalk) has been previously observed by singlemolecule FRET (8,9) and cryo-EM (33). Idling of the empty ribosome along the same path has also been inferred from a series of X-ray structures (34).…”

Section: Discussionmentioning

confidence: 75%

“…In some cases, however, snapshots of major ribosomal regions with large conformational flexibility have defied classification into discrete states altogether, even by the most advanced analytical methods (7). Single-molecule FRET experiments have yielded evidence for discrete conformational changes in single, freely equilibrating pretranslocational ribosomes, and provided ensemble averages for such changes, but have been unable to provide data for short-lived intermediates (8,9).…”

mentioning

confidence: 99%

Trajectories of the ribosome as a Brownian nanomachine

Dashti

Schwander

Langlois

et al. 2014

223

222

A Brownian machine, a tiny device buffeted by the random motions of molecules in the environment, is capable of exploiting these thermal motions for many of the conformational changes in its work cycle. Such machines are now thought to be ubiquitous, with the ribosome, a molecular machine responsible for protein synthesis, increasingly regarded as prototypical. Here we present a new analytical approach capable of determining the free-energy landscape and the continuous trajectories of molecular machines from a large number of snapshots obtained by cryogenic electron microscopy. We demonstrate this approach in the context of experimental cryogenic electron microscope images of a large ensemble of nontranslating ribosomes purified from yeast cells. The freeenergy landscape is seen to contain a closed path of low energy, along which the ribosome exhibits conformational changes known to be associated with the elongation cycle. Our approach allows model-free quantitative analysis of the degrees of freedom and the energy landscape underlying continuous conformational changes in nanomachines, including those important for biological function.cryo-electron microscopy | elongation cycle | manifold embedding | nanomachines | translation

“…However, bulk FRET studies suggested that hybrid states coupled to the ratchet-like motion may form spontaneously, i.e., without binding EF-G (9,10). Further evidence for spontaneous ratcheting was provided by single-molecule FRET studies, which showed individual pretranslocation ribosomes oscillating between the nonrotated and the rotated states (4). Also the tRNAs were observed to fluctuate between classic and hybrid states in dynamic equilibrium (11), possibly sampling through additional intermediate states.…”

mentioning

confidence: 99%

Structure of ratcheted ribosomes with tRNAs in hybrid states

Julián

Konevega

Scheres³

et al. 2008

167

176

During protein synthesis, tRNAs and mRNA move through the ribosome between aminoacyl (A), peptidyl (P), and exit (E) sites of the ribosome in a process called translocation. Translocation is accompanied by the displacement of the tRNAs on the large ribosomal subunit toward the hybrid A/P and P/E states and by a rotational movement (ratchet) of the ribosomal subunits relative to one another. So far, the structure of the ratcheted state has been observed only when translation factors were bound to the ribosome. Using cryo-electron microscopy and classification, we show here that ribosomes can spontaneously adopt a ratcheted conformation with tRNAs in their hybrid states. The peptidyl-tRNA molecule in the A/P state, which is visualized here, is not distorted compared with the A/A state except for slight adjustments of its acceptor end, suggesting that the displacement of the A-site tRNA on the 50S subunit is passive and is induced by the 30S subunit rotation. Simultaneous subunit ratchet and formation of the tRNA hybrid states precede and may promote the subsequent rapid and coordinated tRNA translocation on the 30S subunit catalyzed by elongation factor G.translocation ͉ elongation factor G ͉ cryo-electron microscopy D uring the elongation cycle, the tRNAs⅐mRNA complex is translocated to allow reading of the following codon on mRNA. Translocation is promoted by elongation factor G (EF-G) and accompanied by GTP hydrolysis. During translocation, the ribosome changes from the pretranslocation state with deacylated tRNA in the peptidyl site (P site) and peptidyltRNA in the aminoacyl site (A site) to the posttranslocation state where A-and P-site tRNAs have moved to P and exit (E) sites, respectively. The universal design of ribosomes from two subunits inspired early suggestions that translocation may involve a movement of the subunits relative to each other (1, 2). Such models imply the existence of intermediate states for the tRNAs that differ from the classic A, P, and E states. Chemical probing experiments with pretranslocation ribosomes indicated that after peptidyl transfer the acceptor ends of the tRNAs spontaneously moved on the large 50S subunit toward their posttranslocation positions (3), indicating that peptidyl-tRNA and deacylated tRNA entered hybrid A/P and P/E states, respectively. The transition was observed in the absence of EF-G, and the driving force for the movement was attributed to different affinities of the A, P, and E sites on the 50S subunit (4) for the chemically different acceptor ends of the tRNAs (5, 6). Nevertheless, cryo-electron microscopy (cryoEM) of ribosomes in the pretranslocation state showed the tRNAs in their classic A, P, and E states (7). Although this result was difficult to reconcile with the hybrid-state model, it is important to note that in that complex the occupancy of the E site by deacylated tRNA may have prevented the P-site tRNA to enter the P/E-site hybrid state.CryoEM of ribosomes in complex with EF-G revealed a relative rotation between subunits referred to as ratc...