Trajectories of the ribosome as a Brownian nanomachine

Abstract

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

Functional analysis of the uL11 protein impact on translational machinery

“…41 The pivotal role in trGTPase binding and stimulation has been attributed to the GAC region composed of an rRNA fragment called a sarcinricin loop (SRL) and a protein element, i.e. an oligomeric Pstalk structure.…”

The Linac Coherent Light Source: Recent Developments and Future Plans

“…The high repetition rate of LCLS-II in the near-optimum tender X-ray range can potentially generate 10 8 to 10 10 scattering snapshots per day, thereby driving a qualitative advance in our ability to map the conformational energy landscapes traversed by biological nanomachines. Cryogenic electron microscopy (cryo-EM) results demonstrate the potential to extract three-dimensional structure [67], conformational movies, and energy landscapes from ultralow-signal snapshots of biological complexes cryo-trapped in random orientations at statistically determined points in their work cycle [68]. In the low-signal regime, the number of available snapshots ultimately determines the information content of a conformational movie and the detail with which an energy landscape can be mapped.…”

The potential use of single-particle electron microscopy as a tool for structure-based inhibitor design

“…This opens up new avenues for the structural determination of previously intractable targets, from large macromolecules such as viruses to membrane proteins, where the presence of detergent makes crystallization a challenge (Bill et al, 2011). EM also offers potential advantages for drug discovery by being able to trap different conformational states of a protein, moving away from static crystal structures to a dynamic ensemble of different states (Dashti et al, 2014;Frank & Ourmazd, 2016). This could prove to be extremely powerful, as inhibitorbinding pockets can display plasticity and change shape as the protein samples different conformations.…”

“…Work is ongoing to develop time-resolved EM through a variety of methods, including rapid mixing/spraying in order to physically trap the protein of interest in a variety of functional states (Walker et al, 1995;Lu et al, 2009;Chen & Frank, 2016), as well as computational manifold mapping approaches, which have already been utilized to identify several functional conformational states of the ribosome (Dashti et al, 2014;Frank & Ourmazd, 2016). By removing the constraints of a crystal lattice, it is possible to map the larger conformational changes associated with catalytic cycling, which is invaluable in understanding the full dynamic range of a protein-protein complex.…”