Two-Dimensional Crystallisation of Membrane Proteins and Structural Assessment

Two-dimensional (2D) crystallisation of Membrane proteins reconstitutes them into their native environment, the lipid bilayer. Electron crystallography allows the structural analysis of these regular protein–lipid arrays up to atomic resolution. The crystal quality depends on the protein purity, ist stability and on the crystallisation conditions. The basics of 2D crystallisation and different recent advances are reviewed and electron crystallography approaches summarised. Progress in 2D crystallisation, sample preparation, image detectors and automation of the data acquisition and processing pipeline makes 2D electron crystallography particularly attractive for the structural analysis of membrane proteins that are too small for single-particle analyses and too unstable to form three-dimensional (3D) crystals.

Functional analysis of p53 phosphorylation and a p53 hot spot mutation

The tumor suppressor p53 is a phosphoprotein which functions as a transcriptional activator. By monitoring the transcriptional activity, we studied how p53 functions is regulated in relation to cell growth and contact inhibition. When cells were arrested at G1 phase of the cell cycle by contact inhibition, we found that p53 transactivation function was suppressed. When contact inhibition was overridden by cyclin E overexpression which stimulates cell cycle progression, p53 function was restored. This observation led to the development of a cell density assay to study the regulation of p53 function during cell cycle for the functional significance of p53 phosphorylation. The murine p53 is phosphorylated at serines 7, 9, 12, 18, 37, 312 and 389. To understand the role of p53 phosphorylation, we generated p53 constructs encoding serine-to-alanine or serine-to-glutamate mutations at these codons. The transcriptional activity were measured in cells capable of contact inhibition. In low-density cycling cells, no difference in transcriptional activity was found between wild type p53 and any of the mutants. In contact-inhibited cells, however, only mutations of p53 at serine 389 resulted in altered responses to cell cycle arrest and to cyclin E overexpression. The mutant with serine-to-glutamate substitution at codon 389 retained its function in contact inhibited cells. Cyclin E overexpression in these cells induced p53 phosphorylation at serine 389. Furthermore, we showed that phosphorylation at serine 389 regulates p53 DNA binding activity. Our findings implicate that phosphorylation is an important mechanism for p53 activation.^ p53 is the most frequently mutated gene in human tumors. To study the mechanism of p53 inactivation by mutations, we carried out detailed analysis of a murine p53 mutation with an arginine-to-tryptophane substitution at codon 245. The corresponding human p53 mutation at amino acid 248 is the most frequently mutated codon in tumors. We showed that this mutant is inactive in suppressing focus formation, binding to DNA and transactivation. Structural analysis revealed that this mutant assumes the wild type protein conformation. These findings define a novel class of p53 mutations and help to understand structure-function relationship of p53. ^

Analysis of the railway track as a spatially periodic structure

This article presents a new and computationally efficient method of analysis of a railway track modelled as a continuous beam of 2N spans supported by elastic vertical springs. The main feature of this method is its important reduction in computational effort with respect to standard matrix methods of structural analysis. In this article, the whole structure is considered to be a repetition of a single one. The analysis presented is applied to a simple railway track model, i.e. to a repetitive beam supported on vertical springs (sleepers). The proposed method of analysis is based on the general theory of spatially periodic structures. The main feature of this theory is the possibility to apply Discrete Fourier Transform (DFT) in order to reduce a large system of q(2N + 1) linear stiffness equilibrium equations to a set of 2N + 1 uncoupled systems of q equations each. In this way, a dramatic reduction of the computational effort of solving the large system of equations is achieved. This fact is particularly important in the analysis of railway track structures, in which N is a very large number (around several thousands), and q = 2, the vertical displacement and rotation, is very small. The proposed method allows us to easily obtain the exact solution given by Samartín [1], i.e. the continuous beam railway track response. The comparison between the proposed method and other methods of analysis of railway tracks, such as Lorente de Nó and Zimmermann-Timoshenko, clearly shows the accuracy of the obtained results for the proposed method, even for low values of N. In addition, identical results between the proposed and the Lorente methods have been found, although the proposed method seems to be of simpler application and computationally more efficient than the Lorente one. Small but significative differences occur between these two methods and the one developed by Zimmermann-Timoshenko. This article also presents a detailed sensitivity analysis of the vertical displacement of the sleepers. Although standard matrix methods of structural analysis can handle this railway model, one of the objectives of this article is to show the efficiency of DFT method with respect to standard matrix structural analysis. A comparative analysis between standard matrix structural analysis and the proposed method (DFT), in terms of computational time, input, output and also software programming, will be carried out. Finally, a URL link to a MatLab computer program list, based on the proposed method, is given

Numerical analysis of shell and spatial structures

Since the advent of the computer into the engineering field, the application of the numerical methods to the solution of engineering problems has grown very rapidly. Among the different computer methods of structural analysis the Finite Element (FEM) has been predominantly used. Shells and space structures are very attractive and have been constructed to solve a large variety of functional problems (roofs, industrial building, aqueducts, reservoirs, footings etc). In this type of structures aesthetics, structural efficiency and concept play a very important role. This class of structures can be divided into three main groups, namely continuous (concrete) shells, space frames and tension (fabric, pneumatic, cable etc )structures. In the following only the current applications of the FEM to the analysis of continuous shell structures will be discussed. However, some of the comments on this class of shells can be also applied to some extend to the others, but obviously specific computational problems will be restricted to the continuous shells. Different aspects, such as, the type of elements,input-output computational techniques etc, of the analysis of shells by the FEM will be described below. Clearly, the improvements and developments occurring in general for the FEM since its first appearance in the fifties have had a significative impact on the particular class of structures under discussion.

Numerical methods in Nonlinear Analysis of shell structures

The design of shell and spatial structures represents an important challenge even with the use of the modern computer technology.If we concentrate in the concrete shell structures many problems must be faced,such as the conceptual and structural disposition, optimal shape design, analysis, construction methods, details etc. and all these problems are interconnected among them. As an example the shape optimization requires the use of several disciplines like structural analysis, sensitivity analysis, optimization strategies and geometrical design concepts. Similar comments can be applied to other space structures such as steel trusses with single or double shape and tension structures. In relation to the analysis the Finite Element Method appears to be the most extended and versatile technique used in the practice. In the application of this method several issues arise. First the derivation of the pertinent shell theory or alternatively the degenerated 3-D solid approach should be chosen. According to the previous election the suitable FE model has to be adopted i.e. the displacement,stress or mixed formulated element. The good behavior of the shell structures under dead loads that are carried out towards the supports by mainly compressive stresses is impaired by the high imperfection sensitivity usually exhibited by these structures. This last effect is important particularly if large deformation and material nonlinearities of the shell may interact unfavorably, as can be the case for thin reinforced shells. In this respect the study of the stability of the shell represents a compulsory step in the analysis. Therefore there are currently very active fields of research such as the different descriptions of consistent nonlinear shell models given by Simo, Fox and Rifai, Mantzenmiller and Buchter and Ramm among others, the consistent formulation of efficient tangent stiffness as the one presented by Ortiz and Schweizerhof and Wringgers, with application to concrete shells exhibiting creep behavior given by Scordelis and coworkers; and finally the development of numerical techniques needed to trace the nonlinear response of the structure. The objective of this paper is concentrated in the last research aspect i.e. in the presentation of a state-of-the-art on the existing solution techniques for nonlinear analysis of structures. In this presentation the following excellent reviews on this subject will be mainly used.

Analysis of single and binary phases in cerium doped sodium bismuth titanate -Na0.5Bi0.5TiO3 materials

The pure and cerium doped sodium bismuth titanate inorganic powders were synthesized by solid state reaction method. The presence of rhombohedral phase was observed in cerium doped NBT compounds. At 1200 ºC, the 5% of cerium doped NBT compound forms single perovskite phase. The samples of x = 0.10 and 0.15 were heat treated to 1350 ºC, the binary phases with cerium and bismuth oxides were observed. The X-ray diffraction, fourier transform infrared spectroscopy, reflectance spectra, differential thermal analysis and thermo gravimetric analysis were used to analyze the various properties of samples. Moreover, the effects of cerium doping and calcining temperature on NBT samples were investigated. In this work we present our recent results on the synthesis and characterization of Ce doped sodium bismuth titanate materials.

Funicularity and equilibrium for high-performance conceptual structural design

Las estructuras que trabajan por forma se caracterizan por la íntima e indisociable relación entre geometría y comportamiento estructural. Por consiguiente, la elección de una apropiada geometría es el paso previo indispensable en el diseño conceptual de dichas estructuras. En esa tarea, la selección de las posibles geometrías antifuniculares para las distribuciones de cargas permanentes más habituales son más bien limitadas y, muchas veces, son criterios no estructurales (adaptabilidad funcional, estética, proceso constructivo, etc.) los que no permiten la utilización de dichas geometrías que garantizarían el máximo aprovechamiento del material. En este contexto, esta tesis estudia la posibilidad de obtener una estructura sin momentos flectores incluso si la geometría no es antifunicular para sus cargas permanentes. En efecto, esta tesis presenta un procedimiento, basado en la estática gráfica, que demuestra cómo un conjunto de cargas adicionales, introducidas a través de un sistema de pretensado exterior con elementos post-tesos, puede eliminar los momentos flectores debidos a cargas permanentes en cualquier geometría plana. Esto se traduce en una estructura antifunicular que proporciona respuestas innovadoras a demandas conjuntas de versatilidad arquitectónica y optimización del material. Dicha metodología gráfica ha sido implementada en un software distribuido libremente (EXOEQUILIBRIUM), donde el análisis estructural y la variación geométrica están incluidos en el mismo entorno interactivo y paramétrico. La utilización de estas herramientas permite más versatilidad en la búsqueda de nuevas formas eficientes, lo cual tiene gran importancia en el diseño conceptual de estructuras, liberando al ingeniero de la limitación del propio cálculo y de la incomprensión del comportamiento estructural, facilitando extraordinariamente el hecho creativo a la luz de una metodología de este estilo. Esta tesis incluye la aplicación de estos procedimientos a estructuras de cualquier geometría y distribución inicial de cargas, así como el estudio de diferentes posibles criterios de diseño para optimizar la posición del sistema de post-tesado. Además, la metodología ha sido empleada en el proyecto de maquetas a escala reducida y en la construcción de un pabellón hecho enteramente de cartón, lo que ha permitido obtener una validación física del procedimiento desarrollado. En definitiva, esta tesis expande de manera relevante el rango de posibles geometrías antifuniculares y abre enormes posibilidades para el diseño de estructuras que combinan eficiencia estructural y flexibilidad arquitectónica.Curved structures are characterized by the critical relationship between their geometry and structural behaviour, and selecting an appropriate shape in the conceptual design of such structures is important for achieving materialefficiency. However, the set of bending-free geometries are limited and, often, non-structural design criteria (e.g., usability, architectural needs, aesthetics) prohibit the selection of purely funicular or antifunicular shapes. In response to this issue, this thesis studies the possibility of achieving an axial-only behaviour even if the geometry departs from the ideally bending-free shape. This dissertation presents a new design approach, based on graphic statics that shows how bending moments in a two-dimensional geometry can be eliminated by adding forces through an external post-tensioning system. This results in bending-free structures that provide innovative answers to combined demands on versatility and material optimization. The graphical procedure has been implemented in a free-downloadable design-driven software (EXOEQUILIBRIUM) where structural performance evaluations and geometric variation are embedded within an interactive and parametric working environment. This provides greater versatility in finding new efficient structural configurations during the first design stages, bridging the gap between architectural shaping and structural analysis. The thesis includes the application of the developed graphical procedure to shapes with random curvature and distribution of loads. Furthermore, the effect of different design criteria on the internal force distribution has been analyzed. Finally, the construction of reduced- and large-scale models provides further physical validation of the method and insights about the structural behaviour of these structures. In summary, this work strongly expands the range of possible forms that exhibit a bending-free behaviour and, de facto, opens up new possibilities for designs that combine high-performing solutions with architectural freedom.

Structural homologies with ATP- and folate-binding enzymes in the crystal structure of folylpolyglutamate synthetase

Folylpolyglutamate synthetase, which is responsible for the addition of a polyglutamate tail to folate and folate derivatives, is an ATP-dependent enzyme isolated from eukaryotic and bacterial sources, where it plays a key role in the retention of the intracellular folate pool. Here, we report the 2.4-Å resolution crystal structure of the MgATP complex of the enzyme from Lactobacillus casei. The structural analysis reveals that folylpolyglutamate synthetase is a modular protein consisting of two domains, one with a typical mononucleotide-binding fold and the other strikingly similar to the folate-binding enzyme dihydrofolate reductase. We have located the active site of the enzyme in a large interdomain cleft adjacent to an ATP-binding P-loop motif. Opposite this site, in the C domain, a cavity likely to be the folate binding site has been identified, and inspection of this cavity and the surrounding protein structure suggests that the glutamate tail of the substrate may project into the active site. A further feature of the structure is a well defined Ω loop, which contributes both to the active site and to interdomain interactions. The determination of the structure of this enzyme represents the first step toward the elucidation of the molecular mechanism of polyglutamylation of folates and antifolates.

STRESS (structural engineering system solver) a computer programming system for structural engineering problems.

Mode of access: Internet.

A study of matrix analysis methods for inelastic structures. Project no. 1467, Task no. 146701, prepared under Contract no. AF 33 (657)-8717 by Massachusetts Institute of Technology, Aeroelastic and Structures Research Laboratory.

Cover title.

Computational methods for structural mechanics and dynamics : proceedings of a workshop ... held at NASA Langley Research Center, Hampton, Virginia, June 19-21, 1985 /

Includes bibliographical references.

Strength and elasticity of structural members /

Mode of access: Internet.

Nonparametric Identication and Structural Estimation of Auction Models

Thesis (Ph.D.)--University of Washington, 2016-06

Molecular analysis of the pathway for the synthesis of thiol tripeptides in the model legume Lotus japonicus

The thiol tripeptides, glutathione (GSH) and homoglutathione (hGSH), perform multiple roles in legumes, including protection against toxicity of free radicals and heavy metals. The three genes involved in the synthesis of GSH and hGSH in the model legume, Lotus japonicus, have been fully characterized and appear to be present as single copies in the genome. The gamma-glutamylcysteine synthetase (gammaecs) gene was mapped on the long arm of chromosome 4 (70.0 centimorgans [cM]) and consists of 15 exons, whereas the glutathione synthetase (gshs) and homoglutathione synthetase (hgshs) genes were mapped on the long arm of chromosome 1 (81.3 cM) and found to be arranged in tandem, with a separation of approximately 8 kb. Both genes consist of 12 exons of exactly the same size (except exon 1, which is similar). Two types of transcripts were detected for the gshs gene, which putatively encode proteins localized in the plastids and cytosol. Promoter regions contain cis-acting regulatory elements that may be involved in the plant's response to light, hormones, and stress. Determination of transcript levels, enzyme activities, and thiol contents in nodules, roots, and leaves revealed that gammaecs and hgshs are expressed in all three plant organs, whereas gshs is significantly functional only in nodules. This strongly suggests an important role of GSH in the rhizobia-legume symbiosis.

Twists, knots, and rings in proteins: Structural definition of the cyclotide framework

In recent years an increasing number of miniproteins containing an amide-cyclized backbone have been discovered. The cyclotide family is the largest group of such proteins and is characterized by a circular protein backbone and six conserved cysteine residues linked by disulfide bonds in a tight core of the molecule. These form a cystine knot in which an embedded ring formed by two of the disulfide bonds and the connecting backbone segment is threaded by a third disulfide bond. In the current study we have undertaken high resolution structural analysis of two prototypic cyclotides, kalata B1 and cycloviolacin O1, to define the role of the conserved residues in the sequence. We provide the first comprehensive analysis of the topological features in this unique family of proteins, namely rings (a circular backbone), twists (a cis-peptide bond in the Mobius cyclotides) and knots (a knotted arrangement of the disulfide bonds).