Pilar Ferrer

Crystal Structures of Bacterial Peptidoglycan Amidase Ampd and an Unprecedented Activation Mechanism.

AmpD is a cytoplasmic peptidoglycan (PG) amidase involved in bacterial cell-wall recycling and in induction of β-lactamase, a key enzyme of β-lactam antibiotic resistance. AmpD belongs to the amidase_2 family that includes zinc-dependent amidases and the peptidoglycan-recognition proteins (PGRPs), highly conserved pattern-recognition molecules of the immune system. Crystal structures of Citrobacter freundii AmpD were solved in this study for the apoenzyme, for the holoenzyme at two different pH values, and for the complex with the reaction products, providing insights into the PG recognition and the catalytic process. These structures are significantly different compared with the previously reported NMR structure for the same protein. The NMR structure does not possess an accessible active site and shows the protein in what is proposed herein as an inactive “closed” conformation. The transition of the protein from this inactive conformation to the active “open” conformation, as seen in the x-ray structures, was studied by targeted molecular dynamics simulations, which revealed large conformational rearrangements (as much as 17 Å) in four specific regions representing one-third of the entire protein. It is proposed that the large conformational change that would take the inactive NMR structure to the active x-ray structure represents an unprecedented mechanism for activation of AmpD. Analysis is presented to argue that this activation mechanism might be representative of a regulatory process for other intracellular members of the bacterial amidase_2 family of enzymes.

Size determination and quantification of engineered cerium oxide nanoparticles by flow field-flow fractionation coupled to inductively coupled plasma mass spectrometry

Facing the lack of studies on characterization and quantification of cerium oxide nanoparticles (CeO2 NPs), whose consumption and release is greatly increasing, this work proposes a method for their sizing and quantification by Flow Field-flow Fractionation (FFFF) coupled to Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). Two modalities of FFFF (Asymmetric Flow- and Hollow Fiber-Flow Field Flow Fractionation, AF4 and HF5, respectively) are compared, and their advantages and limitations discussed. Experimental conditions (carrier composition, pH, ionic strength, crossflow and carrier flow rates) are studied in detail in terms of NP separation, recovery, and repeatability. Size characterization of CeO2 NPs was addressed by different approaches. In the absence of feasible size standards of CeO2 NPs, suspensions of Ag, Au, and SiO2 NPs of known size were investigated. Ag and Au NPs failed to show a comparable behavior to that of the CeO2 NPs, whereas the use of SiO2 NPs provided size estimations in agreement to those predicted by the theory. The latter approach was thus used for characterizing the size of CeO2 NPs in a commercial suspension. Results were in adequate concordance with those achieved by transmission electron microscopy, X-ray diffraction and dynamic light scattering. The quantification of CeO2 NPs in the commercial suspension by AF4-ICP-MS required the use of a CeO2 NPs standards, since the use of ionic cerium resulted in low recoveries (99 ± 9% vs. 73 ± 7%, respectively). A limit of detection of 0.9 μg L(-1) CeO2 corresponding to a number concentration of 1.8 × 1012 L(-1) for NPs of 5 nm was achieved for an injection volume of 100 μL.

Thickness evolution of the twin structure and shear strain in LSMO films

X-ray diffraction analysis and orientation contrast scanning electron microscopy imaging of La0.7Sr0.3MnO3 epitaxial layers grown on (001)-SrTiO3 substrates have been used to track the shear strain and twin domain period as a function of the thickness of the films, t. To this end, the diffraction by a periodically modulated twinned structure is analyzed in detail. In contrast with current equilibrium models, here we demonstrate the occurrence of a critical thickness, tτ ∼ 2.0–2.5 nm, for twin formation in rhombohedral perovskite films. The absence of twinning below tτ is explained by the formation of a monoclinic interfacial phase presumably driven by electronic interactions between film and substrate not taken into account in theoretical models. Above tτ, twin domains develop concomitantly with the build-up of misfit shear strains associated with the formation of the rhombohedral structure. At a thickness ∼10 nm, the in-plane and out-of-plane shear strain components exhibit similar values, as imposed by the rhombohedral symmetry. However, upon increasing the film thickness, both strain components are found to follow divergent trajectories indicating a progressive perturbation of the octahedral framework which allows the in-plane lattice parameters to remain fully strained within the explored thickness range (up to 475 nm). Despite these structural perturbations, the twin size follows a t1/2 dependence as predicted for homogeneous films by equilibrium models.

Environmental influence on Zn-histidine complexes under no-packing conditions

This paper describes a combined structural analysis of the Zn-histidine complex, using two different and complementary techniques, X-ray absorption spectroscopy (XAS) and surface X-ray diffraction, paying special attention to the environmental conditions. The current procedure for investigating macromolecules consists of examining simple molecules that exhibit properties similar to those of the larger ones, whose functionality is totally related to the atomic structure. The detailed study of the bonding structure formed by zinc and histidine amino acids is motivated by the fact that this material serves as a model for metalloproteins, such as in metalloproteinase, acting as active sites in enzymatic or structural functions. For XAS modeling, Zn-histidine complexes were dissolved in several aqueous solutions, over a wide pH range. Correlations among the degree of protonation, the steric impediment, and the multiple combinations of the histidine amino acid have been found. For the diffraction study, high-quality crystals grown by the seeding method in a supersaturated solution have been studied, and the samples for the surface diffraction study were mounted on a cell specially designed for solid-liquid or solid-gas interface analysis. The surface structural model was built from XAS results. In both cases, the obtained structures are compared with the bulk one, showing atomic differences and highlighting the importance of the environment in which the complex is studied.

Correlation between the electronic and atomic structure, transport properties, and oxygen vacancies on La0.7Ca0.3MnO3 thin films

We present a study of the role of oxygen vacancies on the atomic and electronic structure and transport properties on a 20 nm thick La0.7Ca0.3MnO3 film grown by the pulsed laser deposition method on a SrTiO3 (001) substrate. The results show that oxygen vacancies induce an atomic structure modification characterized by the movement of the La/Ca cations to the perovskite regular position, by the reduction of the MnO6 basal plane rotation, and by a cooperative tilting of the octahedra along the out-of-plane direction. The out-of-plane lattice parameter increases due to the reduction of the Mn valence upon oxygen vacancies creation. As a consequence, a shift of the Metal-to-Insulator transition to lower temperatures is found to occur. We discuss the influence of the competitive phenomena of manganese valence and Mn-O-Mn bond distortion on the transport properties of manganite thin films.

A double crystal X-ray monochromator for the SpLine diffraction and absorption synchrotron bending magnet beamline at the ESRF

The CRG BM25-SpLine Beamline is located at bending magnet 25 of the ESRF. The beamline, which is split in two branches, is devoted to XAS, XRD and HAXPES. The photon energy covered by both branches range between 5 and 45 keV. The beamline double crystal monochromator (DCM) uses two parallel Si(111) crystals in (+, −) configuration to produce a monochromatic exit beam parallel to the incident white X-ray beam. It accepts 2mrad of radiation from the bending magnet. The DCM has been recently upgraded. Several special features concerning the cooling of the first crystal, second crystal positioning and sagittal focusing mechanism has been improved. In this work a detailed description of the performed modifications is presented.

Multi-use high/low-temperature and pressure compatible portable chamber for in situ grazing-incidence X-ray scattering studies.

The multipurpose portable ultra-high-vacuum-compatible chamber described in detail in this article has been designed to carry out grazing-incidence X-ray scattering techniques on the BM25-SpLine CRG beamline at the ESRF. The chamber has a cylindrical form, built on a 360° beryllium double-ended conflate flange (CF) nipple. The main advantage of this chamber design is the wide sample temperature range, which may be varied between 60 and 1000 K. Other advantages of using a cylinder are that the wall thickness is reduced to a minimum value, keeping maximal solid angle accessibility and keeping wall absorption of the incoming X-ray beam constant. The heat exchanger is a customized compact liquid-nitrogen (LN2) continuous-flow cryostat. LN2 is transferred from a storage Dewar through a vacuum-isolated transfer line to the heat exchanger. The sample is mounted on a molybdenum support on the heat exchanger, which is equipped with a BORALECTRIC heater element. The chamber versatility extends to the operating pressure, ranging from ultra-high vacuum (<10(-10) mbar) to high pressure (up to 3 × 10(3) mbar). In addition, it is equipped with several CF ports to allocate auxiliary components such as capillary gas-inlet, viewports, leak valves, ion gun, turbo pump, etc., responding to a large variety of experiment requirements. A movable slits set-up has been foreseen to reduce the background and diffuse scattering produced at the beryllium wall. Diffraction data can be recorded either with a point detector or with a bi-dimensional CCD detector, or both detectors simultaneously. The system has been designed to carry out a multitude of experiments in a large variety of environments. The system feasibility is demonstrated by showing temperature-dependence grazing-incidence X-ray diffraction and conductivity measurements on a 20 nm-thick La0.7Ca0.3MnO3 thin film grown on a SrTiO3(001) substrate.

A portable powder-liquid high corrosion-resistant reaction cell for in situ X-ray diffraction and absorption studies of heterogeneous powder-liquid reactions and phase transformations

A portable powder-liquid high corrosion-resistant reaction cell has been designed to follow in situ reactions by X-ray powder diffraction and X-ray absorption spectroscopy techniques in transmission mode. The cell has been conceived to be mounted on the experimental stations for diffraction and absorption of the Spanish CRG SpLine-BM25 beamline at the ESRF. In the case of the diffraction technique, data can be collected with either a point detector or a two-dimensional CCD detector. Using the 2D-CCD camera, the cell can be used for time-resolved in situ studies of phase transitions and reactions. Powder reactants and/or products are kept at a fixed position in a vertical geometry in the X-ray pathway, which is minimized in order to reduce the X-ray absorption by the reaction bath. Sample is fixed by a porous membrane under forced liquid reflux circulation, assuring total powder-liquid contact, with an accurate temperature control in the range from 20 to 220°C.