Dual-axis STEM tomography at 200 kV: Setup, performance, limitations.

The interpretation of cell biological processes hinges on the elucidation of the underlying structures. Their three-dimensional analysis using electron tomography has extended our understanding of cellular organelles tremendously. The investigations depend on the availability of appropriate instruments for data recording. So far, such investigations have been done to a great extent on 300 keV transmission electron microscopes. Here we show the implementation of STEM tomography on a 200 kV FEG transmission electron microscope, including the tuning of the condenser for forming a beam with a small illumination aperture, dual-axis data recording, and evaluation of the maximum sample thickness and quality of the data. Our results show that the approach is accomplishable and promising, with high reliability, and reaching excellent data quality from plastic sections with a thickness of at least 900 nm.

Structural basis of highly conserved ribosome recycling in eukaryotes and archaea.

Ribosome-driven protein biosynthesis is comprised of four phases: initiation, elongation, termination and recycling. In bacteria, ribosome recycling requires ribosome recycling factor and elongation factor G, and several structures of bacterial recycling complexes have been determined. In the eukaryotic and archaeal kingdoms, however, recycling involves the ABC-type ATPase ABCE1 and little is known about its structural basis. Here we present cryo-electron microscopy reconstructions of eukaryotic and archaeal ribosome recycling complexes containing ABCE1 and the termination factor paralogue Pelota. These structures reveal the overall binding mode of ABCE1 to be similar to canonical translation factors. Moreover, the iron-sulphur cluster domain of ABCE1 interacts with and stabilizes Pelota in a conformation that reaches towards the peptidyl transferase centre, thus explaining how ABCE1 may stimulate peptide-release activity of canonical termination factors. Using the mechanochemical properties of ABCE1, a conserved mechanism in archaea and eukaryotes is suggested that couples translation termination to recycling, and eventually to re-initiation.