Visualization of the Hybrid State of tRNA Binding Promoted by Spontaneous Ratcheting of the Ribosome

Agirrezabala, Xabier; Lei, Jianlin; Brunelle, Julie L.; Ortiz‐Meoz, Rodrigo F.; Green, Rachel; Frank, Joachim

doi:10.1016/j.molcel.2008.10.001

Cited by 236 publications

(202 citation statements)

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“…In 10% of the vacant 70S ribosomes, the small ribosomal subunit was rotated by 8 relative to the large subunit when compared to the majority (90%) of 70S ribosomes that were observed in the non-rotated conformation. The rotated vacant 70S ribosome reconstructed by Chen et al is very similar in conformation to rotated ribosomes containing tRNAs bound in the hybrid (A/P and P/E) states that were previously visualized by cryo-EM and X-ray crystallography (Agirrezabala et al, 2008;Dunkle et al, 2011;Juliá n et al, 2008). Chen et al also found that the non-rotated vacant 70S ribosomes were composed of two distinct structural classes, which differed in the degree of rotation of the head domain of the 30S ribosomal subunit relative to the rest of the 30S subunit.…”

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Ribosome Subunit Joining Frozen in Time

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Ermolenko

2015

Structure

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Ribosome Subunit Joining Frozen in Time

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Ermolenko

2015

Structure

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“…Based on the results of the smFRET studies of the PRE ribosome, it could be expected that cryo-EM of this kind of sample would yield strongly heterogeneous data. Indeed, reference-based classification, where density maps of rotated and unrotated ribosomes were used as references, showed a clear division of the data into two subpopulations [57,58] which, after separate reconstruction, yielded unambiguous evidence of the two states-namely unrotated along with classical A/A-, P/P-tRNA positions, and rotated with hybrid A/P, P/E positions.…”

Section: Evidence Of Intersubunit Rotation In the Absence Of Elongatimentioning

confidence: 99%

The translation elongation cycle—capturing multiple states by cryo-electron microscopy

Frank

2017

Phil. Trans. R. Soc. B

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One contribution of 11 to a theme issue 'Perspectives on the ribosome'. During the work cycle of elongation, the ribosome, a molecular machine of vast complexity, exists in a large number of states distinguished by constellation of its subunits, its subunit domains and binding partners. Single-particle cryogenic electron microscopy (cryo-EM), developed over the past 40 years, is uniquely suited to determine the structure of molecular machines in their native states. With the emergence, 10 years ago, of unsupervised clustering techniques in the analysis of single-particle data, it has been possible to determine multiple structures from a sample containing ribosomes equilibrating in different thermally accessible states. In addition, recent advances in detector technology have made it possible to reach near-atomic resolution for some of these states. With these capabilities, single-particle cryo-EM has been at the forefront of exploring ribosome dynamics during its functional cycle, along with single-molecule fluorescence resonance energy transfer and molecular dynamics computations, offering insights into molecular architecture uniquely honed by evolution to capitalize on thermal energy in the ambient environment.This article is part of the themed issue 'Perspectives on the ribosome'.

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“…We early recognized that this movement is facilitated by a ratchetlike reorganization of the ribosome [15]. More recently we discovered [16] that the ribosome at the point after peptide bond formation exists in multiple conformational states characterized by different intersubunit rotations and tRNA binding configurations. Thanks to the above-mentioned development of novel classification software these states could be extracted and independently reconstructed [17].…”

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Exploring the Dynamics of Supramolecular Machines With Cryo-Electron Microscopy

Frank¹

2014

New Chemistry and New Opportunities From the Expanding Protein Universe

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Present state of researchSupramolecular machines perform their work in the cell by going through many different states, distinguished by different conformations and free-energy levels. Ideally, in order to find out how these machines work, we would create a suitable in vitro environment containing all components including energy supply that allows the machine to function. We would then aim to take a "movie," capturing their structure at highest resolution in a continuous fashion. Keeping within that film analogy, we might consider taking a large number of "snapshots" in equal small time intervals, each short enough, as in the macroscopic world, to eliminate jarring transitions. However, we would find out that this project has flaws both on the conceptual and the practical level. Conceptually, it is incorrect to equate a molecular machine's progress to the workings of a macroscopic machine in motion since the states are not ordered in sequence of time but are visited in a stochastic manner, with occasional irreversible events such as NTP hydrolysis as the only mark of progress. In practical terms, there are in fact two problems, one affecting the way data for any given state can be captured, the other affecting the ability to obtain coverage of states in a continuum.First of all, the visualization of a structure requires some form of radiation which imparts energy on the molecule and changes it in the very process. Minimization of these damaging interactions calls for a radiation dose so low that averaging over a sufficiently large population is required for visualization. This requirement, in turn, translates into a complicated way each snapshot must be taken: the structural information in every state has to be gathered from many different copies of the molecule. While in forming such an average, crystallographic approaches -X-ray and electron crystallography -are able to take advantage of the regular arrangement of molecules in a crystal, single-particle electron microscopy must first determine the precise orientation of each molecule from its projection image [1]. The second hurdle interfering with the idea of making a movie is that only a limited number of states are sufficiently populated to allow a three-dimensional structure to be determined. Thus the "movie frames" in between these states remain empty, undetermined.New Chemistry and New Opportunities from the Expanding Protein Universe Downloaded from www.worldscientific.com by RUTGERS UNIVERSITY on 08/17/15. For personal use only.

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Visualization of the Hybrid State of tRNA Binding Promoted by Spontaneous Ratcheting of the Ribosome

Cited by 236 publications

References 56 publications

Ribosome Subunit Joining Frozen in Time

Ribosome Subunit Joining Frozen in Time

The translation elongation cycle—capturing multiple states by cryo-electron microscopy

Exploring the Dynamics of Supramolecular Machines With Cryo-Electron Microscopy

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