[en] Cryo electron tomography (cryo-ET) can provide cellular and molecular structural information on various biological samples. However, the detailed interpretation of tomograms reconstructed from single-tilt data tends to suffer from low signal-to-noise ratio and artefacts caused by some systematically missing angular views. While these can be overcome by sub-tomogram averaging, they remain limiting for the analysis of unique structures. Double-tilt ET can improve the tomogram quality by acquiring a second tilt series after an in-plane rotation, but its usage is not widespread yet because it is considered technically demanding and it is rarely used under cryo conditions. Here we show that double-tilt cryo-ET improves the quality of 3D reconstructions so significantly that even single particle analysis can be envisaged despite of the intrinsically low image contrast obtained from frozen-hydrated specimens. This is illustrated by the analysis of eukaryotic polyribosomes in which individual ribosomes were reconstructed using single-tilt, partial and full double-tilt geometries. The improved tomograms favour the faster convergence of iterative sub-tomogram averaging and allow a better 3D classification using multivariate statistical analysis. Our study of single particles and molecular assemblies within polysomes illustrates that the dual-axis approach is particularly useful for cryo applications of ET, both for unique objects and for structures that can be classified and averaged. - Highlights: ► Double-tilt cryo-ET improves 3D reconstructions thus making single particle analysis possible. ► Dual-axis cryo-ET data favour a faster convergence of iterative sub-tomogram averaging. ► Individual ribosomes were reconstructed from single-tilt, partial/ full double-tilt geometries. ► Double-tilt cryo-ET facilitates analysis of larger molecular assemblies such as in cell sections. ► Dual-axis cryo-ET is applicable to unique objects and to structures that can be classified and averaged
Journal
[en] Highlights: • Cryo-electron microscopy of Salivary Gland Hypertrophy Virus was performed. • The major structural features of the SGHV virus from tsetse flies were determined. • The envelope and nucleocapsid of the SGHV particles are both helically organized. Glossina palipides salivary gland hypertrophy virus (GpSGHV) infects tsetse flies, which are vectors for African trypanosomosis. This virus represents a major challenge in insect mass rearing and has hampered the implementation of the sterile insect technique programs in the Member States of the International Atomic Energy Agency. GpSGHV virions consist of long rod-shaped particles over 9000 Å in length, but little is known about their detailed structural organization. We show by cryo electron microscopy and cryo electron tomography that the GpSGHV virion has a unique, non-icosahedral helical structure. Its envelope exhibits regularly spaced spikes that protrude from the lipid bilayer and are arranged on a four-start helix. This study provides a detailed insight into the 3D architecture of GpSGHV, which will help to understand the viral life cycle and possibly allow the design of antiviral strategies in the context of tsetse fly infections.
Journal
[en] The crystal structures of the eubacterial translation initiation factor 2 in apo form and with bound GDP and GTP reveal conformational changes upon nucleotide binding and hydrolysis, notably of the catalytically important histidine in the switch II region. Translation initiation factor 2 (IF2) is involved in the early steps of bacterial protein synthesis. It promotes the stabilization of the initiator tRNA on the 30S initiation complex (IC) and triggers GTP hydrolysis upon ribosomal subunit joining. While the structure of an archaeal homologue (a/eIF5B) is known, there are significant sequence and functional differences in eubacterial IF2, while the trimeric eukaryotic IF2 is completely unrelated. Here, the crystal structure of the apo IF2 protein core from Thermus thermophilus has been determined by MAD phasing and the structures of GTP and GDP complexes were also obtained. The IF2–GTP complex was trapped by soaking with GTP in the cryoprotectant. The structures revealed conformational changes of the protein upon nucleotide binding, in particular in the P-loop region, which extend to the functionally relevant switch II region. The latter carries a catalytically important and conserved histidine residue which is observed in different conformations in the GTP and GDP complexes. Overall, this work provides the first crystal structure of a eubacterial IF2 and suggests that activation of GTP hydrolysis may occur by a conformational repositioning of the histidine residue
Journal
Acta Crystallographica. Section D: Biological Crystallography; ISSN 0907-4449; 
; CODEN ABCRE6; v. 69(Pt 6); p. 925-933
; CODEN ABCRE6; v. 69(Pt 6); p. 925-933