Specific interaction between EF-G and RRF and its implication for GTP-dependent ribosome splitting into subunits

After termination of protein synthesis, the bacterial ribosome is split into its 30S and 50S subunits by the action of ribosome recycling factor (RRF) and elongation factor G (EF-G) in a guanosine 5′-triphosphate (GTP)-hydrolysis-dependent manner. Based on a previous cryo-electron microscopy study of ribosomal complexes, we have proposed that the binding of EF-G to an RRF-containing posttermination ribosome triggers an interdomain rotation of RRF, which destabilizes two strong intersubunit bridges (B2a and B3) and, ultimately, separates the two subunits. Here, we present a 9-Å (Fourier shell correlation cutoff of 0.5) cryo-electron microscopy map of a 50S·EF-G·guanosine 5′-[(βγ)-imido]triphosphate·RRF complex and a quasi-atomic model derived from it, showing the interaction between EF-G and RRF on the 50S subunit in the presence of the noncleavable GTP analogue guanosine 5′-[(βγ)-imido]triphosphate. The detailed information in this model and a comparative analysis of EF-G structures in various nucleotide- and ribosome-bound states show how rotation of the RRF head domain may be triggered by various domains of EF-G. For validation of our structural model, all known mutations in EF-G and RRF that relate to ribosome recycling have been taken into account. More importantly, our results indicate a substantial conformational change in the Switch I region of EF-G, suggesting that a conformational signal transduction mechanism, similar to that employed in transfer RNA translocation on the ribosome by EF-G, translates a large-scale movement of EF-G's domain IV, induced by GTP hydrolysis, into the domain rotation of RRF that eventually splits the ribosome into subunits.     

Refinement of comparative models of protein structure by using multicanonical molecular dynamics simulations

Comparative modelling is a powerful method that easily predicts a considerably accurate structure of a protein by using a template structure having a similar amino-acid sequence to the target protein. However, in the region where the amino-acid sequence is different between the target and the template, the predicted structure remains unreliable. In such a case, the model has to be refined. In the present study, we explored the possibility of a molecular dynamics-based method, using the human SAP Src Homology 2 (SH2) domain as the modelling target. The multicanonical method was used to alleviate the multiple-minima problem and the generalised Born/surface area model was used to reduce the computational cost. In addition, position restraints were imposed on the atoms in the reliable regions to avoid unnecessary conformational sampling. We analyzed the conformational distribution of the ligand-recognition loop of the domain and found that the most populated conformational clusters in the ensemble of the model agreed well with one of the two major clusters in the ensemble of the reference simulation starting from the crystal structure. This demonstrates that the current refinement method can significantly improve the accuracy of an unreliable region in a comparative model.     

Opinion Practical and ethical considerations for students conducting ecological research involving wildlife

This commentary outlines relevant changes to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes for ecologists and students active in field‐based wildlife research. All field‐based activities that require the approval of an Animal Ethics Committee (AEC) have now been detailed within the revised Code of Practice. Researchers and their students are required to give explicit consideration to the humane principles of replacement, reduction and refinement within their experimental protocols as a part of the AEC permission procedure. University‐based researchers also have obligations to train graduate students in the ethical as well as the practical components of wildlife research. In promoting this educational aim, a summary of ethical considerations in wildlife research is offered as a basis for discussion: the regulatory framework of research animal welfare (including an introduction to the Code of Practice, the AEC process and some thoughts on experimental design); and the humane principles on which the Code of Practice is based. Some practical considerations for field work relevant to students’ research training are also described.     

Tightening the Crosslinking Distance Restraints for Better Resolution of Protein Structure and Dynamics

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Driven to near-experimental accuracy by refinement via molecular dynamics simulations

Protein model refinement has been an essential part of successful protein structure prediction. Molecular dynamics simulation-based refinement methods have shown consistent improvement of protein models. There had been progress in the extent of refinement for a few years since the idea of ensemble averaging of sampled conformations emerged. There was little progress in CASP12 because conformational sampling was not sufficiently diverse due to harmonic restraints. During CASP13, a new refinement method was tested that achieved significant improvements over CASP12. The new method intended to address previous bottlenecks in the refinement problem by introducing new features. Flat-bottom harmonic restraints replaced harmonic restraints, sampling was performed iteratively, and a new scoring function and selection criteria were used. The new protocol expanded conformational sampling at reduced computational costs. In addition to overall improvements, some models were refined significantly to near-experimental accuracy.     

A novel protocol of energy optimisation for predicted protein structures built by homology modelling

Homology modelling was applied to predict the three-dimensional (3D) structures of six sets of lipase proteins. Sequence identities between the target and template were 34.6, 44.9, 57.4, 69.9, 79.0 and 86.2%, respectively. Then, eight different protocols including three optimising factors [periodically bounded cell (PBC) water, molecular dynamics (MD) simulation, ‘grade-unpacking’ strategy or ‘combinatorial’ strategy] were used to refine the initial model of each system. By comparing the energy-optimised models with the true 3D structure of the target protein in terms of all backbone atoms' root mean square deviation, we determined a novel but all-purpose protocol for model refinement. The protocol refined a homology model by adopting the ‘grade-unpacking’ strategy for energy minimisation while the model was solvated in PBC water. Furthermore, by comparing the influence of each single optimising factor on the accuracy of the refined structure, we found that introducing the MD simulation into the model refinement method would decrease the accuracy of the final protein structure while methods with either PBC water or the ‘grade-unpacking’ strategy would increase the accuracy of the final model.     

goCTF: Geometrically optimized CTF determination for single-particle cryo-EM

Preferred particle orientation represents a recurring problem in single-particle cryogenic electron microcopy (cryo-EM). A specimen-independent approach through tilting has been attempted to increase particle orientation coverage, thus minimizing anisotropic three-dimensional (3D) reconstruction. However, focus gradient is a critical issue hindering tilt applications from being a general practice in single-particle cryo-EM. The present study describes a newly developed geometrically optimized approach, goCTF, to reliably determine the global focus gradient. A novel strategy of determining contrast transfer function (CTF) parameters from a sector of the signal preserved power spectrum is applied to increase reliability. Subsequently, per-particle based local focus refinement is conducted in an iterative manner to further improve the defocus accuracy. Novel diagnosis methods using a standard deviation defocus plot and goodness of fit heatmap have also been proposed to evaluate CTF fitting quality prior to 3D refinement. In a benchmark study, goCTF processed a published single-particle cryo-EM dataset for influenza hemagglutinin trimer collected at a 40-degree specimen tilt. The resulting 3D reconstruction map was improved from 4.1?Å to 3.7?Å resolution. The goCTF program is built on the open-source code of CTFFIND4, which adopts a consistent user interface for ease of use.