Assessment of interatomic potentials for atomistic analysis of static and dynamic properties of screw dislocations in W

Screw dislocations in bcc metals display non-planar cores at zero temperature which result in high lattice friction and thermally-activated strain rate behavior. In bcc W, electronic structure molecular statics calculations reveal a compact, non-degenerate core with an associated Peierls stress between 1.7 and 2.8 GPa. However, a full picture of the dynamic behavior of dislocations can only be gained by using more efficient atomistic simulations based on semiempirical interatomic potentials. In this paper we assess the suitability of five different potentials in terms of static properties relevant to screw dislocations in pure W. Moreover, we perform molecular dynamics simulations of stress-assisted glide using all five potentials to study the dynamic behavior of screw dislocations under shear stress. Dislocations are seen to display thermally-activated motion in most of the applied stress range, with a gradual transition to a viscous damping regime at high stresses. We find that one potential predicts a core transformation from compact to dissociated at finite temperature that affects the energetics of kink-pair production and impacts the mechanism of motion. We conclude that a modified embedded-atom potential achieves the best compromise in terms of static and dynamic screw dislocation properties, although at an expense of about ten-fold compared to central potentials.

Reliability issues and cost structure in the 13000 SHS rural electrification programme in Morocco

Operalion and mainlenance (O&M) casi 01 solar home syslems (SHS) in pholovollaie rural electrification (PVRE) programmes is a key factor in the economic viability and long lerm suslainability. This paper reports on a 13000 inslalled SHSs programme carried oul in Moroceo. Il is presenled Ihe reliability analysis 01 Ihe SHSs as well as the distribution of real cests of installation, O&M and management to oblain respeclively Ihe reliability lunelions 01 Ihe SHS componenls and lo eharaclerize the overall programme cast structure.

Analysis of the static and dynamic behaviour of hydraulic fills

En la actualidad existe un gran conocimiento en la caracterización de rellenos hidráulicos, tanto en su caracterización estática, como dinámica. Sin embargo, son escasos en la literatura estudios más generales y globales de estos materiales, muy relacionados con sus usos y principales problemáticas en obras portuarias y mineras. Los procedimientos semi‐empíricos para la evaluación del efecto silo en las celdas de cajones portuarios, así como para el potencial de licuefacción de estos suelos durantes cargas instantáneas y terremotos, se basan en estudios donde la influencia de los parámetros que los rigen no se conocen en gran medida, dando lugar a resultados con considerable dispersión. Este es el caso, por ejemplo, de los daños notificados por el grupo de investigación del Puerto de Barcelona, la rotura de los cajones portuarios en el Puerto de Barcelona en 2007. Por estos motivos y otros, se ha decidido desarrollar un análisis para la evaluación de estos problemas mediante la propuesta de una metodología teórico‐numérica y empírica. El enfoque teórico‐numérico desarrollado en el presente estudio se centra en la determinación del marco teórico y las herramientas numéricas capaces de solventar los retos que presentan estos problemas. La complejidad del problema procede de varios aspectos fundamentales: el comportamiento no lineal de los suelos poco confinados o flojos en procesos de consolidación por preso propio; su alto potencial de licuefacción; la caracterización hidromecánica de los contactos entre estructuras y suelo (camino preferencial para el flujo de agua y consolidación lateral); el punto de partida de los problemas con un estado de tensiones efectivas prácticamente nulo. En cuanto al enfoque experimental, se ha propuesto una metodología de laboratorio muy sencilla para la caracterización hidromecánica del suelo y las interfaces, sin la necesidad de usar complejos aparatos de laboratorio o procedimientos excesivamente complicados. Este trabajo incluye por tanto un breve repaso a los aspectos relacionados con la ejecución de los rellenos hidráulicos, sus usos principales y los fenómenos relacionados, con el fin de establecer un punto de partida para el presente estudio. Este repaso abarca desde la evolución de las ecuaciones de consolidación tradicionales (Terzaghi, 1943), (Gibson, English & Hussey, 1967) y las metodologías de cálculo (Townsend & McVay, 1990) (Fredlund, Donaldson and Gitirana, 2009) hasta las contribuciones en relación al efecto silo (Ranssen, 1985) (Ravenet, 1977) y sobre el fenómeno de la licuefacción (Casagrande, 1936) (Castro, 1969) (Been & Jefferies, 1985) (Pastor & Zienkiewicz, 1986). Con motivo de este estudio se ha desarrollado exclusivamente un código basado en el método de los elementos finitos (MEF) empleando el programa MATLAB. Para ello, se ha esablecido un marco teórico (Biot, 1941) (Zienkiewicz & Shiomi, 1984) (Segura & Caron, 2004) y numérico (Zienkiewicz & Taylor, 1989) (Huerta & Rodríguez, 1992) (Segura & Carol, 2008) para resolver problemas de consolidación multidimensional con condiciones de contorno friccionales, y los correspondientes modelos constitutivos (Pastor & Zienkiewicz, 1986) (Fiu & Liu, 2011). Asimismo, se ha desarrollado una metodología experimental a través de una serie de ensayos de laboratorio para la calibración de los modelos constitutivos y de la caracterización de parámetros índice y de flujo (Castro, 1969) (Bahda 1997) (Been & Jefferies, 2006). Para ello se han empleado arenas de Hostun como material (relleno hidráulico) de referencia. Como principal aportación se incluyen una serie de nuevos ensayos de corte directo para la caracterización hidromecánica de la interfaz suelo – estructura de hormigón, para diferentes tipos de encofrados y rugosidades. Finalmente, se han diseñado una serie de algoritmos específicos para la resolución del set de ecuaciones diferenciales de gobierno que definen este problema. Estos algoritmos son de gran importancia en este problema para tratar el procesamiento transitorio de la consolidación de los rellenos hidráulicos, y de otros efectos relacionados con su implementación en celdas de cajones, como el efecto silo y la licuefacciones autoinducida. Para ello, se ha establecido un modelo 2D axisimétrico, con formulación acoplada u‐p para elementos continuos y elementos interfaz (de espesor cero), que tratan de simular las condiciones de estos rellenos hidráulicos cuando se colocan en las celdas portuarias. Este caso de estudio hace referencia clara a materiales granulares en estado inicial muy suelto y con escasas tensiones efectivas, es decir, con prácticamente todas las sobrepresiones ocasionadas por el proceso de autoconsolidación (por peso propio). Por todo ello se requiere de algoritmos numéricos específicos, así como de modelos constitutivos particulares, para los elementos del continuo y para los elementos interfaz. En el caso de la simulación de diferentes procedimientos de puesta en obra de los rellenos se ha requerido la modificacion de los algoritmos empleados para poder así representar numéricamente la puesta en obra de estos materiales, además de poder realizar una comparativa de los resultados para los distintos procedimientos. La constante actualización de los parámetros del suelo, hace también de este algoritmo una potente herramienta que permite establecer un interesante juego de perfiles de variables, tales como la densidad, el índice de huecos, la fracción de sólidos, el exceso de presiones, y tensiones y deformaciones. En definitiva, el modelo otorga un mejor entendimiento del efecto silo, término comúnmente usado para definir el fenómeno transitorio del gradiente de presiones laterales en las estructuras de contención en forma de silo. Finalmente se incluyen una serie de comparativas entre los resultados del modelo y de diferentes estudios de la literatura técnica, tanto para el fenómeno de las consolidaciones por preso propio (Fredlund, Donaldson & Gitirana, 2009) como para el estudio del efecto silo (Puertos del Estado, 2006, EuroCódigo (2006), Japan Tech, Stands. (2009), etc.). Para concluir, se propone el diseño de un prototipo de columna de decantación con paredes friccionales, como principal propuesta de futura línea de investigación. Wide research is nowadays available on the characterization of hydraulic fills in terms of either static or dynamic behavior. However, reported comprehensive analyses of these soils when meant for port or mining works are scarce. Moreover, the semi‐empirical procedures for assessing the silo effect on cells in floating caissons, and the liquefaction potential of these soils during sudden loads or earthquakes are based on studies where the underlying influence parameters are not well known, yielding results with significant scatter. This is the case, for instance, of hazards reported by the Barcelona Liquefaction working group, with the failure of harbor walls in 2007. By virtue of this, a complex approach has been undertaken to evaluate the problem by a proposal of numerical and laboratory methodology. Within a theoretical and numerical scope, the study is focused on the numerical tools capable to face the different challenges of this problem. The complexity is manifold; the highly non‐linear behavior of consolidating soft soils; their potentially liquefactable nature, the significance of the hydromechanics of the soil‐structure contact, the discontinuities as preferential paths for water flow, setting “negligible” effective stresses as initial conditions. Within an experimental scope, a straightforward laboratory methodology is introduced for the hydromechanical characterization of the soil and the interface without the need of complex laboratory devices or cumbersome procedures. Therefore, this study includes a brief overview of the hydraulic filling execution, main uses (land reclamation, filled cells, tailing dams, etc.) and the underlying phenomena (self‐weight consolidation, silo effect, liquefaction, etc.). It comprises from the evolution of the traditional consolidation equations (Terzaghi, 1943), (Gibson, English, & Hussey, 1967) and solving methodologies (Townsend & McVay, 1990) (Fredlund, Donaldson and Gitirana, 2009) to the contributions in terms of silo effect (Ranssen, 1895) (Ravenet, 1977) and liquefaction phenomena (Casagrande, 1936) (Castro, 1969) (Been & Jefferies, 1985) (Pastor & Zienkiewicz, 1986). The novelty of the study lies on the development of a Finite Element Method (FEM) code, exclusively formulated for this problem. Subsequently, a theoretical (Biot, 1941) (Zienkiewicz and Shiomi, 1984) (Segura and Carol, 2004) and numerical approach (Zienkiewicz and Taylor, 1989) (Huerta, A. & Rodriguez, A., 1992) (Segura, J.M. & Carol, I., 2008) is introduced for multidimensional consolidation problems with frictional contacts and the corresponding constitutive models (Pastor & Zienkiewicz, 1986) (Fu & Liu, 2011). An experimental methodology is presented for the laboratory test and material characterization (Castro 1969) (Bahda 1997) (Been & Jefferies 2006) using Hostun sands as reference hydraulic fill. A series of singular interaction shear tests for the interface calibration is included. Finally, a specific model algorithm for the solution of the set of differential equations governing the problem is presented. The process of consolidation and settlements involves a comprehensive simulation of the transient process of decantation and the build‐up of the silo effect in cells and certain phenomena related to self‐compaction and liquefaction. For this, an implementation of a 2D axi‐syimmetric coupled model with continuum and interface elements, aimed at simulating conditions and self‐weight consolidation of hydraulic fills once placed into floating caisson cells or close to retaining structures. This basically concerns a loose granular soil with a negligible initial effective stress level at the onset of the process. The implementation requires a specific numerical algorithm as well as specific constitutive models for both the continuum and the interface elements. The simulation of implementation procedures for the fills has required the modification of the algorithm so that a numerical representation of these procedures is carried out. A comparison of the results for the different procedures is interesting for the global analysis. Furthermore, the continuous updating of the model provides an insightful logging of variable profiles such as density, void ratio and solid fraction profiles, total and excess pore pressure, stresses and strains. This will lead to a better understanding of complex phenomena such as the transient gradient in lateral pressures due to silo effect in saturated soils. Interesting model and literature comparisons for the self‐weight consolidation (Fredlund, Donaldson, & Gitirana, 2009) and the silo effect results (Puertos del Estado (2006), EuroCode (2006), Japan Tech, Stands. (2009)). This study closes with the design of a decantation column prototype with frictional walls as the main future line of research.

Beam Spectral Element with FRP Debonding

The behaviour of the interface between the FRP and the concrete is the key factor controlling debonding failures in FRP-strengthened RC structures. This defect can cause reductions in static strength, structural integrity and the change in the dynamic behavior of the structure. The adverse effect on the dynamic behavior of the defects can be utilized as an effective means for identifying and assessing both the location and size of debonding at its earliest stages. The presence of debonding changes the structural dynamic characteristics and might be traced in modal parameters, dynamic strain and wave patterns etc. Detection of minor local defects, as those origin of a future debonding, requires working at high frequencies so that the wavelength of the excited is small and sensitive enough to detect local damage. The development of a spectral element method gives a large potential in high-frequency structural modeling. In contrast to the conventional finite element, since inertial properties are modeled exactly few elements are necessary to capture very accurate solutions at the highest frequencies in large regions. A wide variety of spectral elements have been developed for structural members over finite and semi-infinite regions. The objective of this paper is to develop a Spectral Finite Element Model to efficiently capture the behavior of intermediate debonding of a FRP strengthened RC beam during wave-based diagnostics.

Estudio teórico-experimental para la mejora de la resistencia estructural de autocares basado en el diseño de uniones y asientos

La presente tesis analiza la mejora de la resistencia estructural ante vuelco de autocares enfocando dos vías de actuación: análisis y propuestas de requisitos reglamentarios a nivel europeo y la generación de herramientas que ayuden al diseño y a la verificación de estos requisitos. Los requisitos reglamentarios de resistencia estructural a vuelco contemplan la superestructura de los vehículos pero no para los asientos y sistemas de retención. La influencia de los pasajeros retenidos es superior a la incluida en reglamentación (Reg. 66.01) debiendo considerarse unida al vehículo un porcentaje de la masa de los pasajeros del 91% para cinturón de tres puntos y del 52% para cinturón subabdominal frente al 50% reglamentario para todos los casos. Se ha determinado la cinemática y dinámica del vuelco normativo en sus diferentes fases, formulando las energías en las fases iniciales (hasta el impacto contra el suelo) y determinando la fase final de deformación a través del análisis secuencial de ensayos de módulos reales. Se han determinado los esfuerzos para los asientos que se dividen en dos fases diferenciadas temporalmente: una primera debida a la deformación estructural y una segunda debida al esfuerzo del pasajero retenido que se produce en sentido opuesto (con una deceleración del pasajero en torno a 3.3 g). Se ha caracterizado a través de ensayos cuasi.estáticos el comportamiento de perfiles a flexión y de las uniones estructurales de las principales zonas del vehículo (piso, ventana y techo) verificándose la validez del comportamiento plástico teórico Kecman.García para perfiles de hasta 4 mm de espesor y caracterizando la resistencia y rigidez en la zona elástica de las uniones en función del tipo de refuerzo, materiales y perfiles (análisis de más de 180 probetas). Se ha definido un método de ensayo cuasi.estático para asientos ante esfuerzos de vuelco, ensayándose 19 butacas y determinándose que son resistentes (salvo las uniones a vehículo con pinzas), que son capaces de absorber hasta más de un 17% de la energía absorbida, aunque algunos necesitan optimización para llegar a contribuir en el mecanismo de deformación estructural. Se han generado modelos simplificados para introducir en los modelos barra.rótula plástica: un modelo combinado unión+rótula plástica (que incluye la zona de rigidez determinada en función del tipo de unión) para la superestructura y un modelo simplificado de muelles no.lineales para los asientos. Igualmente se ha generado la metodología de diseño a través de ensayos virtuales con modelos de detalle de elementos finitos tanto de las uniones como de los asientos. Se ha propuesto una metodología de diseño basada en obtener el “mecanismo óptimo de deformación estructural” (elevando la zona de deformación lateral a nivel de ventana y en pilar o en costilla en techo). Para ello se abren dos vías: diseño de la superestructura (selección de perfiles y generación de uniones resistentes) o combinación con asientos (que en lugar de solo resistir las cargas pueden llegar a modificar el mecanismo de deformación). Se ha propuesto una metodología de verificación alternativa al vuelco de vehículo completo que contempla el cálculo cuasi.estático con modelos simplificados barra.rótula plástica más el ensayo de una sección representativa con asientos y utillajes antropomórficos retenidos que permite validar el diseño de las uniones, determinar el porcentaje de energía que debe absorberse por deformación estructural (factor C) y verificar el propio asiento como sistema de retención. ABSTRACT This research analyzes the improvement of the structural strength of buses and coaches under rollover from two perspectives: regulatory requirements at European level and generation of tools that will help to the design and to the verification of requirements. European Regulations about rollover structural strength includes requirements for the superstructure of the vehicles but not about seats, anchorages and restraint systems. The influence of the retained passengers is higher than the one included currently in the Regulations (Reg. 66.01), being needed to consider a 91% of the passenger mass as rigidly joint to the vehicle (for 3 points’ belt, a 52% for 2 points’ belt) instead of the 50% included in the Regulation. Kinematic and dynamic of the normative rollover has been determined from testing of different sections, formulating the energies of the first phases (up to the first impact with the ground) and determining the last deformation phase through sequential analysis of movements and deformations. The efforts due to rollover over the seats have been established, being divided in two different temporal phases: a first one due to the structural deformation of the vehicle and a second one due to the effort of the restrained passenger being this second one in opposite sense (with a passenger deceleration around 3.3 g). From quasi.static testing, the behavior of the structural tubes under flexural loads, including the principal joints in the vehicle (floor, window and roof), the validity of the theoretical plastic behavior according Kecman.García theories have been verified up to 4 mm of thickness. Strength of the joints as well as the stiffness of the elastic zone has been determined in function of main parameters: type of reinforcement, materials and section of the tubes (more than 180 test specimens). It has been defined a quasi.static testing methodology to characterize the seats and restrain system behavior under rollover, testing 19 double seats and concluding that they are resistant (excepting clamping joints), that they can absorb more than a 17 of the absorbed energy, and that some of them need optimization to contribute in the structural deformation mechanism. It has been generated simplified MEF models, to analyze in a beam.plastic hinge model: a combined model joint+plastic hinge (including the stiffness depending on the type of joint) for the superstructure and a simplified model with non.lineal springs to represent the seats. It has been detailed methodologies for detailed design of joints and seats from virtual testing (MEF models). A design methodology based in the “optimized structural deformation mechanism” (increasing the height of deformation of the lateral up to window level) is proposed. Two possibilities are analyzed: design of the superstructure based on the selection of profiles and design of strength joints (were seats only resist the efforts and contribute in the energy absorption) or combination structure.seats, were seats contributes in the deformation mechanism. An alternative methodology to the rollover of a vehicle that includes the quasi.static calculation with simplified models “beam.joint+plastic hinge” plus the testing of a representative section of the vehicle including seats and anthropomorphic ballast restrained by the safety belts is presented. The test of the section allows validate the design of the joints, determine the percentage of energy to be absorbed by structural deformation (factor C) and verify the seat as a retention system.

Probing the Saccharomyces cerevisiae centromeric DNA (CEN DNA)–binding factor 3 (CBF3) kinetochore complex by using atomic force microscopy

Yeast centromeric DNA (CEN DNA) binding factor 3 (CBF3) is a multisubunit protein complex that binds to the essential CDEIII element in CEN DNA. The four CBF3 proteins are required for accurate chromosome segregation and are considered to be core components of the yeast kinetochore. We have examined the structure of the CBF3–CEN DNA complex by atomic force microscopy. Assembly of CBF3–CEN DNA complexes was performed by combining purified CBF3 proteins with a DNA fragment that includes the CEN region from yeast chromosome III. Atomic force microscopy images showed DNA molecules with attached globular bodies. The contour length of the DNA containing the complex is ≈9% shorter than the DNA alone, suggesting some winding of DNA within the complex. The measured location of the single binding site indicates that the complex is located asymmetrically to the right of CDEIII extending away from CDEI and CDEII, which is consistent with previous data. The CEN DNA is bent ≈55° at the site of complex formation. A significant fraction of the complexes are linked in pairs, showing three to four DNA arms, with molecular volumes approximately three times the mean volumes of two-armed complexes. These multi-armed complexes indicate that CBF3 can bind two DNA molecules together in vitro and, thus, may be involved in holding together chromatid pairs during mitosis.

An antagonistic vascular endothelial growth factor (VEGF) variant inhibits VEGF-stimulated receptor autophosphorylation and proliferation of human endothelial cells

Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

Structural basis for chemical inhibition of human blood coagulation factor Xa

Factor Xa, the converting enzyme of prothrombin to thrombin, has emerged as an alternative (to thrombin) target for drug discovery for thromboembolic diseases. An inhibitor has been synthesized and the crystal structure of the complex between Des[1–44] factor Xa and the inhibitor has been determined by crystallographic methods in two different crystal forms to 2.3- and 2.4-Å resolution. The racemic mixture of inhibitor FX-2212, (2RS)-(3′-amidino-3-biphenylyl)-5-(4-pyridylamino)pentanoic acid, inhibits factor Xa activity by 50% at 272 nM in vitro. The S-isomer of FX-2212 (FX-2212a) was found to bind to the active site of factor Xa in both crystal forms. The biphenylamidine of FX-2212a occupies the S1-pocket, and the pyridine ring makes hydrophobic interactions with the factor Xa aryl-binding site. Several water molecules meditate inhibitor binding to residues in the active site. In contrast to the earlier crystal structures of factor Xa, such as those of apo-Des[1–45] factor Xa and Des[1–44] factor Xa in complex with a naphthyl inhibitor DX-9065a, two epidermal growth factor-like domains of factor Xa are well ordered in both our crystal forms as well as the region between the two domains, which recently was found to be the binding site of the effector cell protease receptor-1. This structure provides a basis for designing next generation inhibitors of factor Xa.

A cobalt complex that selectively disrupts the structure and function of zinc fingers

Zinc finger domains are structures that mediate sequence recognition for a large number of DNA-binding proteins. These domains consist of sequences of amino acids containing cysteine and histidine residues tetrahedrally coordinated to a zinc ion. In this report, we present a means to selectively inhibit a zinc finger transcription factor with cobalt(III) Schiff-base complexes. 1H NMR spectroscopy confirmed that the structure of a zinc finger peptide is disrupted by axial ligation of the cobalt(III) complex to the nitrogen of the imidazole ring of a histidine residue. Fluorescence studies reveal that the zinc ion is displaced from the model zinc finger peptide in the presence of the cobalt complex. In addition, gel-shift and filter-binding assays reveal that cobalt complexes inhibit binding of a complete zinc finger protein, human transcription factor Sp1, to its consensus sequence. Finally, a DNA-coupled conjugate of the cobalt complexes selectively inhibited Sp1 in the presence of several other transcription factors.

Crystal structure of the BTB domain from PLZF

The BTB domain (also known as the POZ domain) is an evolutionarily conserved protein–protein interaction motif found at the N terminus of 5–10% of C2H2-type zinc-finger transcription factors, as well as in some actin-associated proteins bearing the kelch motif. Many BTB proteins are transcriptional regulators that mediate gene expression through the control of chromatin conformation. In the human promyelocytic leukemia zinc finger (PLZF) protein, the BTB domain has transcriptional repression activity, directs the protein to a nuclear punctate pattern, and interacts with components of the histone deacetylase complex. The association of the PLZF BTB domain with the histone deacetylase complex provides a mechanism of linking the transcription factor with enzymatic activities that regulate chromatin conformation. The crystal structure of the BTB domain of PLZF was determined at 1.9 Å resolution and reveals a tightly intertwined dimer with an extensive hydrophobic interface. Approximately one-quarter of the monomer surface area is involved in the dimer intermolecular contact. These features are typical of obligate homodimers, and we expect the full-length PLZF protein to exist as a branched transcription factor with two C-terminal DNA-binding regions. A surface-exposed groove lined with conserved amino acids is formed at the dimer interface, suggestive of a peptide-binding site. This groove may represent the site of interaction of the PLZF BTB domain with nuclear corepressors or other nuclear proteins.

Structure of a soluble secreted chemokine inhibitor vCCI (p35) from cowpox virus

Most poxviruses, including variola, the causative agent of smallpox, express a secreted protein of 35 kDa, vCCI, which binds CC-chemokines with high affinity. This viral protein competes with the host cellular CC-chemokine receptors (CCRs), reducing inflammation and interfering with the host immune response. Such proteins or derivatives may have therapeutic uses as anti-inflammatory agents. We have determined the crystal structure to 1.85-Å resolution of vCCI from cowpox virus, the prototype of this poxvirus virulence factor. The molecule is a β-sandwich of topology not previously described. A patch of conserved residues on the exposed face of a β-sheet that is strongly negatively charged might have a role in binding of CC-chemokines, which are positively charged.

Parallel-up structure evidences the molecular directionality during biosynthesis of bacterial cellulose

The “parallel-up” packing in cellulose Iα and Iβ unit cells was experimentally demonstrated by a combination of direct-staining the reducing ends of cellulose chains and microdiffraction-tilting electron crystallographic analysis. Microdiffraction investigation of nascent bacterial cellulose microfibrils showed that the reducing end of the growing cellulose chains points away from the bacterium, and this provides direct evidence that polymerization by the cellulose synthase takes place at the nonreducing end of the growing cellulose chains. This mechanism is likely to be valid also for a number of processive glycosyltransferases such as chitin synthases, hyaluronan synthases, and proteins involved in the synthesis of nodulation factor backbones.

Abrogation of the alternative complement pathway by targeted deletion of murine factor B

To investigate the role of complement protein factor B (Bf) and alternative pathway activity in vivo, and to test the hypothesized potential genetic lethal effect of Bf deficiency, the murine Bf gene was interrupted by exchange of exon 3 through exon 7 (including the factor D cleaving site) with the neor gene. Mice heterozygous for the targeted Bf allele were interbred, yielding Bf-deficient offspring after the F1 generation at a frequency suggesting that Bf deficiency alone has no major effect on fertility or fetal development. However, in the context of one or more genes derived from the 129 mouse strain, offspring homozygous for Bf deficiency were generated at less than expected numbers (P = 0.012). Bf-deficient mice showed no gross phenotypic difference from wild-type littermates. Sera from Bf-deficient mice lacked detectable alternative complement pathway activity; purified mouse Bf overcame the deficit. Classical pathway-dependent total hemolytic activity was lower in Bf-deficient than wild-type mice, possibly reflecting loss of the alternative pathway amplification loop. Lymphoid organ structure and IgG1 antibody response to a T-dependent antigen appeared normal in Bf-deficient mice. Sensitivity to lethal endotoxic shock was not significantly altered in Bf-deficient mice. Thus, deficiency of Bf and alternative complement activation pathway led to a less dramatic phenotype than expected. Nevertheless, these mice provide an excellent model for the assessment of the role of Bf and the alternative pathway in host defense and other functions in vivo.

F-actin and G-actin binding are uncoupled by mutation of conserved tyrosine residues in maize actin depolymerizing factor (ZmADF)

Actin depolymerizing factors (ADF) are stimulus responsive actin cytoskeleton modulating proteins. They bind both monomeric actin (G-actin) and filamentous actin (F-actin) and, under certain conditions, F-actin binding is followed by filament severing. In this paper, using mutant maize ADF3 proteins, we demonstrate that the maize ADF3 binding of F-actin can be spatially distinguished from that of G-actin. One mutant, zmadf3–1, in which Tyr-103 and Ala-104 (equivalent to destrin Tyr-117 and Ala-118) have been replaced by phenylalanine and glycine, respectively, binds more weakly to both G-actin and F-actin compared with maize ADF3. A second mutant, zmadf3–2, in which both Tyr-67 and Tyr-70 are replaced by phenylalanine, shows an affinity for G-actin similar to maize ADF3, but F-actin binding is abolished. The two tyrosines, Tyr-67 and Tyr-70, are in the equivalent position to Tyr-82 and Tyr-85 of destrin, respectively. Using the tertiary structure of destrin, yeast cofilin, and Acanthamoeba actophorin, we discuss the implications of removing the aromatic hydroxyls of Tyr-82 and Tyr-85 (i.e., the effect of substituting phenylalanine for tyrosine) and conclude that Tyr-82 plays a critical role in stabilizing the tertiary structure that is essential for F-actin binding. We propose that this tertiary structure is maintained as a result of a hydrogen bond between the hydroxyl of Tyr-82 and the carbonyl of Tyr-117, which is located in the long α-helix; amino acid components of this helix (Leu-111 to Phe-128) have been implicated in G-actin and F-actin binding. The structures of human destrin and yeast cofilin indicate a hydrogen distance of 2.61 and 2.77 Å, respectively, with corresponding bond angles of 99.5° and 113°, close to the optimum for a strong hydrogen bond.

Structure at 2.7 Å resolution of the Paracoccus denitrificans two-subunit cytochrome c oxidase complexed with an antibody FV fragment

The aa3 type cytochrome c oxidase consisting of the core subunits I and II only was isolated from the soil bacterium Paracoccus denitrificans and crystallized as complex with a monoclonal antibody Fv fragment. Crystals could be grown in the presence of a number of different nonionic detergents. However, only undecyl-β-d-maltoside and cyclohexyl-hexyl-β-d-maltoside yielded well-ordered crystals suitable for high resolution x-ray crystallographic studies. The crystals belong to space group P212121 and diffract x-rays to at least 2.5 Å (1 Å = 0.1 nm) resolution using synchrotron radiation. The structure was determined to a resolution of 2.7 Å using molecular replacement and refined to a crystallographic R-factor of 20.5% (Rfree = 25.9%). The refined model includes subunits I and II and the 2 chains of the Fv fragment, 2 heme A molecules, 3 copper atoms, and 1 Mg/Mn atom, a new metal (Ca) binding site, 52 tentatively identified water molecules, and 9 detergent molecules. Only four of the water molecules are located in the cytoplasmic half of cytochrome c oxidase. Most of them are near the interface of subunits I and II. Several waters form a hydrogen-bonded cluster, including the heme propionates and the Mg/Mn binding site. The Fv fragment binds to the periplasmic polar domain of subunit II and is critically involved in the formation of the crystal lattice. The crystallization procedure is well reproducible and will allow for the analysis of the structures of mechanistically interesting mutant cytochrome c oxidases.