Cryo-electron microscopy as an investigative tool: the ribosome as an example (original) (raw)
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Resolution Revolutions on the Cryo-EM Structures of Ribosome- A Study: 1981-2018
2020
Ribosomes are large ribonucleoprotein complexes that have been proved to be ideal biological sample for the development of the cryo-Electron Microscopy (cryo-EM) field. On the other hand, advancement in cryo-EM technologies have helped in elucidating detailed structure-function aspects of various kinds of ribosomes, belonging to prokaryotic, organellar, and complex eukaryotic systems. In a relatively short duration of time, through rapid technological advances, cryo-EM has generated number of ribosome structures from sub atomic to atomic resolutions with the capability of visualizing the side chain interactions of protein structures. This has given us the scope to visualize various drugs and their binding sites on the ribosomes, wherefrom there are greater chances to improve further and modify the drugs and verify their efficacy. This review provides an account of the improvement of the cryo-EM in the light of performing research on ribosomes and understanding some of the ribosome’s...
Ribosome dynamics: insights from atomic structure modeling into cryo-electron microscopy maps
Annu. Rev. Biophys. Biomol. Struct., 2006
Single-particle cryo-electron microscopy (cryo-EM) is the method of choice for studying the dynamics of macromolecular machines both at a phenomenological and, increasingly, at the molecular level, with the advent of high-resolution component X-ray structures and of progressively improving fitting algorithms. Cryo-EM has shed light on the structure of the ribosome during the four steps of translation: initiation, elongation, termination, and recycling. Interpretation of cryo-EM reconstructions of the ribosome in quasi-atomic detail reveals a picture in which the ribosome uses RNA not only to catalyze chemical reactions, but also as a means for signal transduction over large distances.
2012
Single-particle cryo-electron microscopy (cryo-EM) became a well-established method to study the structure and function of large macromolecular assemblies in a close to physiological environment. Cryo-EM reconstructions of ribosomal complexes trapped at different stages during translation, cotranslational targeting, and translocation provide new insights on a molecular level into these processes, which are vital for the correct localization and folding of all proteins in the cell. The EM structures in combination with biochemical experiments and available highresolution crystal or nuclear magnetic resonance (NMR) structures of individual factors and of the ribosome allow for interpretation in quasi-atomic detail of the molecular mechanism of ribosomal complexes, their conformational changes and dynamic interactions with factors like the signal recognition particle, SRP receptor, the translocon, and the chaperone trigger factor. The snapshots obtained by single-particle EM reconstructions enable us to follow the path of a nascent protein from the peptidyl-transferase center, through the ribosomal tunnel, to and across the translocon in the membrane. With new developments in image processing techniques it is possible to sort a biological homogenous sample into different conformational states and to reach subnanometer resolution such that folding of the nascent chain into secondary structure elements can be directly visualized. With improved cryo-electron tomography and correlative light microscopy and EM, it will be possible to visualize ribosomal complexes in their cellular context.
Structure (London, England : 1993), 2015
Ribosomal subunit association is a key checkpoint in translation initiation but its structural dynamics are poorly understood. Here, we used a recently developed mixing-spraying, time-resolved, cryogenic electron microscopy (cryo-EM) method to study ribosomal subunit association in the sub-second time range. We have improved this method and increased the cryo-EM data yield by tenfold. Pre-equilibrium states of the association reaction were captured by reacting the mixture of ribosomal subunits for 60 ms and 140 ms. We also identified three distinct ribosome conformations in the associated ribosomes. The observed proportions of these conformations are the same in these two time points, suggesting that ribosomes equilibrate among the three conformations within less than 60 ms upon formation. Our results demonstrate that the mixing-spraying method can capture multiple states of macromolecules during a sub-second reaction. Other fast processes, such as translation initiation, decoding, ...
Current Opinion in Structural Biology, 2017
Cryo electron microscopy (cryo-EM) historically has had a strong impact on the structural and mechanistic analysis of protein synthesis by the prokaryotic and eukaryotic ribosomes. Vice versa, studying ribosomes has helped moving forwards many methodological aspects in single particle cryo-EM, at the level of automated data collection and image processing including advanced techniques for particle sorting to address structural and compositional heterogeneity. Here we review some of the latest ribosome structures, where cryo-EM allowed gaining unprecedented insights based on 3D structure sorting with focused classification and refinement methods helping to reach local resolution levels better than 3 Å. Such high-resolution features now enable the analysis of drug interactions with RNA and protein side-chains including even the visualization of chemical modifications of the ribosomal RNA. These advances represent a major breakthrough in structural biology and show the strong potential of cryo-EM beyond the ribosome field including for structure-based drug design.
RNA, 2020
It is only after recent advances in cryo-electron microscopy that it is now possible to describe at high-resolution structures of large macromolecules that do not crystalize. Purified 30S subunits interconvert between an “active” and “inactive” conformation. The active conformation was described by crystallography in the early 2000s, but the structure of the inactive form at high resolution remains unsolved. Here we used cryo-electron microscopy to obtain the structure of the inactive conformation of the 30S subunit to 3.6 Å resolution and study its motions. In the inactive conformation, an alternative base-pairing of three nucleotides causes the region of helix 44, forming the decoding center to adopt an unlatched conformation and the 3′ end of the 16S rRNA positions similarly to the mRNA during translation. Incubation of inactive 30S subunits at 42°C reverts these structural changes. The air–water interface to which ribosome subunits are exposed during sample preparation also peel...
F1000 - Post-publication peer review of the biomedical literature, 2013
Although electron cryo-microscopy (cryo-EM) single-particle analysis has become an important tool for structural biology of large and flexible macro-molecular assemblies, the technique has not yet reached its full potential. Besides fundamental limits imposed by radiation damage, poor detectors and beam-induced sample movement have been shown to degrade attainable resolutions. A new generation of direct electron detectors may ameliorate both effects. Apart from exhibiting improved signal-to-noise performance, these cameras are also fast enough to follow particle movements during electron irradiation. Here, we assess the potentials of this technology for cryo-EM structure determination. Using a newly developed statistical movie processing approach to compensate for beam-induced movement, we show that ribosome reconstructions with unprecedented resolutions may be calculated from almost two orders of magnitude fewer particles than used previously. Therefore, this methodology may expand the scope of high-resolution cryo-EM to a broad range of biological specimens.
Structural Heterogeneities of the Ribosome: New Frontiers and Opportunities for Cryo-EM
Molecules
The extent of ribosomal heterogeneity has caught increasing interest over the past few years, as recent studies have highlighted the presence of structural variations of the ribosome. More precisely, the heterogeneity of the ribosome covers multiple scales, including the dynamical aspects of ribosomal motion at the single particle level, specialization at the cellular and subcellular scale, or evolutionary differences across species. Upon solving the ribosome atomic structure at medium to high resolution, cryogenic electron microscopy (cryo-EM) has enabled investigating all these forms of heterogeneity. In this review, we present some recent advances in quantifying ribosome heterogeneity, with a focus on the conformational and evolutionary variations of the ribosome and their functional implications. These efforts highlight the need for new computational methods and comparative tools, to comprehensively model the continuous conformational transition pathways of the ribosome, as well...