Better Cryo-EM Specimen Preparation: How to Deal with the Air–Water Interface?

Cryogenic electron microscopy (cryo-EM) is now one of the most powerful and widely used methods to determine high-resolution structures of macromolecules. A major bottleneck of cryo-EM is to prepare high-quality vitrified specimen, which still faces many practical challenges. During the conventional vitrification process, macromolecules tend to adsorb at the air–water interface (AWI), which is known unfriendly to biological samples. In this review, we outline the nature of AWI and the problems caused by it, such as unpredictable or uneven particle distribution, protein denaturation, dissociation of complex and preferential orientation. We review and discuss the approaches and underlying mechanisms to deal with AWI: 1) Additives, exemplified by detergents, forming a protective layer at AWI and thus preserving the native folds of target macromolecules. 2) Fast vitrification devices based on the idea to freeze in-solution macromolecules before their touching of AWI. 3) Thin layer of continuous supporting films to adsorb macromolecules, and when functionalized with affinity ligands, to specifically anchor the target particles away from the AWI. Among these supporting films, graphene, together with its derivatives, with negligible background noise and mechanical robustness, has emerged as a new generation of support. These strategies have been proven successful in various cases and enable us a better handling of the problems caused by the AWI in cryo-EM specimen preparation.     

Mechanisms of successive modes of erythrocyte stability and instability in the presence of various polymers

Upon examination in real time of the adhesion of human erythrocytes by observing cells suspended by ultrasonic radiation force in solutions of dextran, polylysine, and polyethylene glycol, it was reported earlier that concave-ended cell pairs and rouleaux are seen in low (0.5–2.0% w/v) concentrations of Dextran T500. At concentrations of 5–7%, dextran spherical cell doublets and convex-ended cell agglutinates are formed. When adhesion occurs in polylysine (MW 14,000) or in polyethylene glycol (MW 8,000) only spherical cell doublets or convex-ended cell clumps occur. The final cell movement completing the formation of these adhesion products takes place over time scales of the order of 1s. In this work, quantitative consideration is given to the extent to which repulsion between adhesion-inducing macromolecules associated with the glycocalyx and those free in solution can influence adhesion through a phase separation effect. It is shown for cells in dextran and in polylysine that the forces associated with this repulsion are of the same order of magnitude as the electrostatic interactions between cells.     

The properties of Bacillus cereus hemolysin II pores depend on environmental conditions

Hemolysin II (HlyII), one of several cytolytic proteins encoded by the opportunistic human pathogen Bacillus cereus, is a member of the family of oligomeric β-barrel pore-forming toxins. This work has studied the pore-forming properties of HlyII using a number of biochemical and biophysical approaches. According to electron microscopy, HlyII protein interacts with liposomes to form ordered heptamer-like macromolecular assemblies with an inner pore diameter of 1.5-2 nm and an outer diameter of 6-8 nm. This is consistent with inner pore diameter obtained from osmotic protection assay. According to the 3D model obtained, seven HlyII monomers might form a pore, the outer size of which has been estimated to be slightly larger than by the other method, with an inner diameter changing from 1 to 4 nm along the channel length. The hemolysis rate has been found to be temperature-dependent, with an explicit lag at lower temperatures. Temperature jump experiments have indicated the pore structures formed at 37 °C and 4 °C to be different. The channels formed by HlyII are anion-selective in lipid bilayers and show a rising conductance as the salt concentration increases. The results presented show for the first time that at high salt concentration HlyII pores demonstrate voltage-induced gating observed at low negative potentials. Taken together we have found that the membrane-binding properties of hemolysin II as well as the properties of its pores strongly depend on environmental conditions. The study of the properties together with structural modeling allows a better understanding of channel functioning.     

Nutrient composition and in vitro ruminal fermentation of tropical legume mixtures with contrasting tannin contents

Various combinations of a low-tannin herbaceous legume (Vigna unguiculata) and foliage of tanniniferous shrub legumes (Calliandra calothyrsus, Flemingia macrophylla and Leucaena leucocephala) or a low-tannin shrub legume (Cratylia argentea), all mixed together with a low-quality tropical grass (Brachiaria humidicola), were tested in vitro for differences in the effects on ruminal fermentation. Two experiments with the gas transducer technique were carried out, where each forage mixture was tested either with or without polyethylene glycol in order to be able to identify tannin-related effects (n = 3). In Experiment 1, a stepwise replacement of V. unguiculata by C. calothyrsus (5:0, 4:1, 3:2, 2:3, 1:4, 0:5) at a legume proportion of 1/3 or 2/3 in the mixture was evaluated. Together with two grass-alone and four pure legume treatments this added up to 30 treatments. In Experiment 2, V. unguiculata was gradually replaced by each of the four shrub legumes (3:0, 2:1, 1:2, 0:3) in grass–legume ratios of 2:1, adding up, together with two grass-alone treatments, to 28 treatments. When added alone, V. unguiculata resulted in high fermentative activity as measured by gas production and kinetics as well as low proportion of undegraded crude protein. When V. unguiculata was replaced by the low-tannin C. argentea in Experiment 2, there was no noticeable difference (P>0.05) in fermentative activity. In both experiments, the effect of the substitution of V. unguiculata by tanniniferous shrub legumes resulted in a declining gas production and an increasing proportion of undegraded crude protein (P<0.001). However, the extent of these changes depended on the level of replacement and the shrub legume species (P<0.001). The results of Experiment 2 illustrate that this was the consequence not only of different tannin contents (less adverse effects with L. leucocephala than with C. calothyrsus) but also differences in the chemical properties of the tannins present in these shrub legume species (much less adverse effects with L. leucocephala than with F. macrophylla despite similar tannin contents). Furthermore these results indicate that, once the extent of the effects of a tanniniferous legume is known, one may calculate the maximal level of replacement of a low-tannin legume in a grass diet possible without negative effects on ruminal fermentation. This allows to improve dry season grass-based diets with as few as possible of the expensive and less well growing low-tannin legume.