Aberration-corrected electron microscopy of nanoparticles

The early history of scanning transmission electron microscopy (STEM) is reviewed as a way to frame the technical issues that make aberration correction an essential upgrade for the study of nanoparticles using STEM. The principles of aberration correction are explained, and the use of aberration-corrected microscopy in the study of nanostructures is exemplified in order to remark the features and challenges in the use of this measuring techniqu

In vivo assembling of bacterial ribosomal protein L11 into yeast ribosomes makes the particles sensitive to the prokaryotic specific antibiotic thiostrepton

Eukaryotic ribosomal stalk protein L12 and its bacterial orthologue L11 play a central role on ribosomal conformational changes during translocation. Deletion of the two genes encoding L12 in Saccharomyces cerevisiae resulted in a very slow-growth phenotype. Gene RPL12B, but not the RPL12A, cloned in centromeric plasmids fully restored control protein level and the growth rate when expressed in a L12-deprived strain. The same strain has been transformed to express Escherichia coli protein EcL11 under the control of yeast RPL12B promoter. The bacterial protein has been found in similar amounts in washed ribosomes from the transformed yeast strain and from control E. coli cells, however, EcL11 was unable to restore the defective acidic protein stalk composition caused by the absence of ScL12 in the yeast ribosome. Protein EcL11 induced a 10% increase in L12-defective cell growth rate, although the in vitro polymerizing capacity of the EcL11-containing ribosomes is restored in a higher proportion, and, moreover, the particles became partially sensitive to the prokaryotic specific antibiotic thiostrepton. Molecular dynamic simulations using modelled complexes support the correct assembly of bacterial L11 into the yeast ribosome and confirm its direct implication of its CTD in the binding of thiostrepton to ribosomes

Tensor Decomposition Reveals Concurrent Evolutionary Convergences and Divergences and Correlations with Structural Motifs in Ribosomal RNA

Evolutionary relationships among organisms are commonly described by using a hierarchy derived from comparisons of ribosomal RNA (rRNA) sequences. We propose that even on the level of a single rRNA molecule, an organism's evolution is composed of multiple pathways due to concurrent forces that act independently upon different rRNA degrees of freedom. Relationships among organisms are then compositions of coexisting pathway-dependent similarities and dissimilarities, which cannot be described by a single hierarchy. We computationally test this hypothesis in comparative analyses of 16S and 23S rRNA sequence alignments by using a tensor decomposition, i.e., a framework for modeling composite data. Each alignment is encoded in a cuboid, i.e., a third-order tensor, where nucleotides, positions and organisms, each represent a degree of freedom. A tensor mode-1 higher-order singular value decomposition (HOSVD) is formulated such that it separates each cuboid into combinations of patterns of nucleotide frequency variation across organisms and positions, i.e., “eigenpositions” and corresponding nucleotide-specific segments of “eigenorganisms,” respectively, independent of a-priori knowledge of the taxonomic groups or rRNA structures. We find, in support of our hypothesis that, first, the significant eigenpositions reveal multiple similarities and dissimilarities among the taxonomic groups. Second, the corresponding eigenorganisms identify insertions or deletions of nucleotides exclusively conserved within the corresponding groups, that map out entire substructures and are enriched in adenosines, unpaired in the rRNA secondary structure, that participate in tertiary structure interactions. This demonstrates that structural motifs involved in rRNA folding and function are evolutionary degrees of freedom. Third, two previously unknown coexisting subgenic relationships between Microsporidia and Archaea are revealed in both the 16S and 23S rRNA alignments, a convergence and a divergence, conferred by insertions and deletions of these motifs, which cannot be described by a single hierarchy. This shows that mode-1 HOSVD modeling of rRNA alignments might be used to computationally predict evolutionary mechanisms

Synthesis of 5-Hydroxyectoine from Ectoine: Crystal Structure of the Non-Heme Iron(II) and 2-Oxoglutarate-Dependent Dioxygenase EctD

As a response to high osmolality, many microorganisms synthesize various types of compatible solutes. These organic osmolytes aid in offsetting the detrimental effects of low water activity on cell physiology. One of these compatible solutes is ectoine. A sub-group of the ectoine producer's enzymatically convert this tetrahydropyrimidine into a hydroxylated derivative, 5-hydroxyectoine. This compound also functions as an effective osmostress protectant and compatible solute but it possesses properties that differ in several aspects from those of ectoine. The enzyme responsible for ectoine hydroxylation (EctD) is a member of the non-heme iron(II)-containing and 2-oxoglutarate-dependent dioxygenases (EC 1.14.11). These enzymes couple the decarboxylation of 2-oxoglutarate with the formation of a high-energy ferryl-oxo intermediate to catalyze the oxidation of the bound organic substrate. We report here the crystal structure of the ectoine hydroxylase EctD from the moderate halophile Virgibacillus salexigens in complex with Fe3+ at a resolution of 1.85 Å. Like other non-heme iron(II) and 2-oxoglutarate dependent dioxygenases, the core of the EctD structure consists of a double-stranded β-helix forming the main portion of the active-site of the enzyme. The positioning of the iron ligand in the active-site of EctD is mediated by an evolutionarily conserved 2-His-1-carboxylate iron-binding motif. The side chains of the three residues forming this iron-binding site protrude into a deep cavity in the EctD structure that also harbours the 2-oxoglutarate co-substrate-binding site. Database searches revealed a widespread occurrence of EctD-type proteins in members of the Bacteria but only in a single representative of the Archaea, the marine crenarchaeon Nitrosopumilus maritimus. The EctD crystal structure reported here can serve as a template to guide further biochemical and structural studies of this biotechnologically interesting enzyme family

Ectoine and hydroxyectoine inhibit aggregation and neurotoxicity of Alzheimer's beta-amyloid

Abstractβ-Amyloid peptide (Aβ) is the major constituent of senile plaques, the key pathological feature of Alzheimer’s disease. Aβ is physiologically produced as a soluble form, but aggregation of Aβ monomers into oligomers/fibrils causes neurotoxic change of the peptide. In nature, many microorganisms accumulate small molecule chaperones (SMCs) under stressful conditions to prevent the misfolding/denaturation of proteins and to maintain their stability. Hence, it is conceivable that SMCs such as ectoine and hydroxyectoine could be potential inhibitors against the aggregate formation of Alzheimer’s Aβ, which has not been studied to date. The current work shows the effectiveness of ectoine and hydroxyectoine on the inhibition of Aβ42 aggregation and toxicity to human neuroblastoma cells. The characterization tools used for this study include thioflavin-T induced fluorescence, atomic force microscopy and cell viability assay. Considering that ectoine and hydroxyectoine are not toxic to cellular environment even at concentrations as high as 100mM, the results may suggest a basis for the development of ectoines as potential inhibitors associated with neurodegenerative diseases