25 research outputs found
A New Model of Chemical Bonding in Ionic Melts
We developed a new physical model to predict macroscopic properties of
inorganic molten systems using a realistic description of inter-atomic
interactions. Unlike the conventional approach, which tends to overestimate
viscosity by several times, our systems consist of a set of ions with an
admixture of neutral atoms. The neutral atom subsystem is a consequence of the
covalent/ionic state reduction, occurring in the liquid phase. Comparison of
the calculated macroscopic properties (shear viscosity and self-diffusion
constants) with the experiment demonstrates good performance of our model. The
presented approach is inspired by a significant degree of covalent interaction
between the alkali and chlorine atoms, predicted by the coupled cluster theory
Impedance analyser error correction using artificial neural networks
The basic difficulties associated with the impedance analyzers design as well as possible their solutions have been outlined in the paper. The article proves advantages of artificial neural networks for correction of frequency errors in impedance measurements. Error correction algorithm for auto-balancing measurement circuit based on neural networks has been developed. Various ways of algorithms implementation on different computing platforms have been considered. The advantages and disadvantages of neural networks vs. classical analytical models have been analyzed. It has been defined that the most promising approach for algorithmic correction based on neural networks are the following cases: impossibility to obtain expressions for correction algorithms analytically; absence of analytical model of measurement channel is given, availability of only experimental data
Structural basis for the inhibition of RecBCD by Gam and its synergistic antibacterial effect with quinolones
Structural basis for DNA strand separation by a hexameric replicative helicase
Hexameric helicases are processive DNA unwinding machines but how they engage with a replication fork during unwinding is unknown. Using electron microscopy and single particle analysis we determined structures of the intact hexameric helicase E1 from papillomavirus and two complexes of E1 bound to a DNA replication fork end-labelled with protein tags. By labelling a DNA replication fork with streptavidin (dsDNA end) and Fab (5′ ssDNA) we located the positions of these labels on the helicase surface, showing that at least 10 bp of dsDNA enter the E1 helicase via a side tunnel. In the currently accepted ‘steric exclusion’ model for dsDNA unwinding, the active 3′ ssDNA strand is pulled through a central tunnel of the helicase motor domain as the dsDNA strands are wedged apart outside the protein assembly. Our structural observations together with nuclease footprinting assays indicate otherwise: strand separation is taking place inside E1 in a chamber above the helicase domain and the 5′ passive ssDNA strands exits the assembly through a separate tunnel opposite to the dsDNA entry point. Our data therefore suggest an alternative to the current general model for DNA unwinding by hexameric helicases
Structure of the hDmc1-ssDNA filament reveals the principles of its architecture
In eukaryotes, meiotic recombination is a major source of genetic diversity, but its defects in humans lead to abnormalities such as Down's, Klinefelter's and other syndromes. Human Dmc1 (hDmc1), a RecA/Rad51 homologue, is a recombinase that plays a crucial role in faithful chromosome segregation during meiosis. The initial step of homologous recombination occurs when hDmc1 forms a filament on single-stranded (ss) DNA. However the structure of this presynaptic complex filament for hDmc1 remains unknown. To compare hDmc1-ssDNA complexes to those known for the RecA/Rad51 family we have obtained electron microscopy (EM) structures of hDmc1-ssDNA nucleoprotein filaments using single particle approach. The EM maps were analysed by docking crystal structures of Dmc1, Rad51, RadA, RecA and DNA. To fully characterise hDmc1-DNA complexes we have analysed their organisation in the presence of Ca2+, Mg2+, ATP, AMP-PNP, ssDNA and dsDNA. The 3D EM structures of the hDmc1-ssDNA filaments allowed us to elucidate the principles of their internal architecture. Similar to the RecA/Rad51 family, hDmc1 forms helical filaments on ssDNA in two states: extended (active) and compressed (inactive). However, in contrast to the RecA/Rad51 family, and the recently reported structure of hDmc1-double stranded (ds) DNA nucleoprotein filaments, the extended (active) state of the hDmc1 filament formed on ssDNA has nine protomers per helical turn, instead of the conventional six, resulting in one protomer covering two nucleotides instead of three. The control reconstruction of the hDmc1-dsDNA filament revealed 6.4 protein subunits per helical turn indicating that the filament organisation varies depending on the DNA templates. Our structural analysis has also revealed that the N-terminal domain of hDmc1 accomplishes its important role in complex formation through domain swapping between adjacent protomers, thus providing a mechanistic basis for coordinated action of hDmc1 protomers during meiotic recombination
Subunit topology in the V type ATPase and related enzymes
During the last decades impressive progress has been made in understanding of the catalytic mechanism of F-type ATP synthase, which is the key enzyme in the energy metabolism of eukaryotes and most bacteria. This enzyme catalyzes the final step in the process of oxidative phosphorylation in bacteria and mitochondria, or in photophosphorylation in chloroplasts. However, some closely related enzymes, the A-type ATP synthase and V-ATPase, have been poorly characterized in terms of structure. These enzymes are unique energy converters in archaea (A-ATPase), bacteria and eukaryotes (V-ATPases). Despite a striking similarity to some of the F-type ATP synthase subunits, the A-type ATP synthase and V-ATPase seem to have a slightly different overall structural organization and regulation. In Chapter I we give an extensive overview of the current knowledge about these enzymes, highlighting the open questions concerning the V-ATPase and A-type ATP synthase structure and subunit interaction. The thesis deals with the structure of the latter two enzymes and their analysis with single particle electron microscopy and a number of other biochemical and biophysical techniques.
Chapter II reports first insight into the subunit composition of the central stalk of the V-ATPase. A Na+-pumping V-ATPase complex from the thermophilic bacterium Caloramator fervidus was isolated. Three subcomplexes with different V1-ATPase subunit composition were purified by anion exchange chromatography following temperature-driven dissociation of the V-ATPase complex. Subsequent analysis of the subcomplexes by polyacrylamide gel electrophoresis revealed the differences in their subunit composition. Single particle electron microscopy was applied for subcomplex analysis. Two-dimensional maps of the averaged subcomplex projections revealed variation in the length of the central stalk. Multivariate statistical analysis of the V1-ATPase projections from the fractions differing in the subunit composition allowed us to reveal the position of the V-ATPase specific subunit C (d in the eukaryotic V-ATPase) on the tip of the central stalk interacting with V0-subunits. Subunit C makes the central stalk of the V-ATPase substantially longer in comparison with the F-ATPase counterpart. Difference mapping was performed to investigate shape and relative position of the stalk subunits in the V-ATPase complex.
In Chapter III we continue the work on the identification of the stalk elements in the V-ATPase complex. An attempt was made to identify a structural analogue of the F-ATPase coupling subunit γ. The experiment consisted of a co-reconstitution of subunits E and G of the yeast V1-ATPase and the α and β subunits of the F1-ATPase from the thermophilic Bacillus PS3 (TF1), and subsequent single particle electron microscopy. The co-reconstitution experiment resulted in an α3β3EG hybrid complex showing 53% of the ATPase activity of TF1. The electron microscopy and single particle image analysis revealed two-dimensional projections of the complex in various positions. An atomic model of the α3β3 subcomplex from TF1 was used for the identification of the V-ATPase subunits by difference mapping. Comparison of the TF1 and α3β3EG hybrid complex revealed that V-ATPase E and G subunits form the central stalk similar to that found in the C. fervidus V1-ATPase projections (Chapter II). On the basis of the biochemical and structural data available, we propose that V-ATPase subunit E is a structural analogue of the F-ATPase subunit γ, while subunit G is located on the periphery of the subcomplex.
In Chapter IV we attempt to identify the position of the Saccharomyces cerevisiae subunit C (Vma5p) in the V1 complex. This subunit is present only in the eukaryotic V-ATPases and is crucial for the regulation of their activity by reversible disassembly. A V1-ATPase hybrid complex was obtained by the reconstitution of the purified Vma5p with the subunit C depleted V1 from Manduca sexta. Because instability of the hybrid complex, a large data set of over 20,000 single particle projections was analyzed. Analysis of single particle projections revealed a rather heterogeneous set of the V1-ATPase complexes. Part of the complexes contained a large external density, which was suggested to represent Vma5p. Comparison of the hybrid complex projections to the averages of the C subunit depleted V1 from M. sexta suggests that, indeed, Vma5p is located on the exterior of the V1-ATPase stalk region. The interaction face of Vma5p with V1 was explored by chemical modification experiments and a model for the binding of Vma5p is presented.
Chapter V describes the first structural characterization by electron microscopy of the intact A-ATP synthase. The A-ATP synthase complex was purified from the archae Methanococcus jannaschii. Two-dimensional projection maps of negatively stained complexes were obtained by electron microscopy and single particle image analysis at a resolution of 1.8 nm. The enzyme with an overall length of 25.9 nm is organized in an A1 headpiece (9.4 x 11.5 nm), and a membrane domain A0 (6.4 x 10.6 nm). Two domains are linked by a stalk region. Similar to the F-ATP synthase and V-ATPases, the central and peripheral stalks were detected. The peripheral stalk moiety is clearly connected to the A0 domain at only one site. In the lower part of the stalk region a horizontal situated rod-like structure (“collar”) was detected. In projection maps it crosses the central stalk density above the contact of the central stalk with A0. The elements of the peripheral stalks extend from the collar up to the top of the A1 headpiece. Superposition of the 3D-reconstruction and the solution structure of the A1 complex from Methanosarcina mazei Gö1 allowed interpretation of the subunit arrangement in the A-ATPase complex.
In Chapter VI the results described in this thesis are summarized and discussed. On the basis of the new structural and biochemical data, a model for the subunit arrangement in the V-ATPase complex is proposed and discussed.
Subunit Composition, Structure, and Distribution of Bacterial V-Type ATPases
The overall structure of V-ATPase complexes resembles that of F-type ATPases, but the stalk region is different and more complex. Database searches followed by sequence analysis of the five water-soluble stalk region subunits C–G revealed that (i) to date V-ATPases are found in 16 bacterial species, (ii) bacterial V-ATPases are closer to archaeal A-ATPases than to eukaryotic V-ATPases, and (iii) different groups of bacterial V-ATPases exist. Inconsistencies in the nomenclature of types and subunits are addressed. Attempts to assign subunit positions in V-ATPases based on biochemical experiments, chemical cross-linking, and electron microscopy are discussed. A structural model for prokaryotic and eukaryotic V-ATPases is proposed. The prokaryotic V-ATPase is considered to have a central stalk between headpiece and membrane flanked by two peripheral stalks. The eukaryotic V-ATPases have one additional peripheral stalk.