Complete structure of the chemosensory array core signalling unit in an E. coli 1 minicell strain

Motile bacteria sense chemical gradients with transmembrane receptors organised in supramolecular signalling arrays. Understanding stimulus detection and transmission at the molecular level requires precise structural characterisation of the array building block known as a core signalling unit. Here we introduce an Escherichia coli strain that forms small minicells possessing extended and highly ordered chemosensory arrays. We use cryo-electron tomography and subtomogram averaging to provide a three-dimensional map of a complete core signalling unit, with visible densities corresponding to the HAMP and periplasmic domains. This map, combined with previously determined high resolution structures and molecular dynamics simulations, yields a molecular model of the transmembrane core signalling unit and enables spatial localisation of its individual domains. Our work thus offers a solid structural basis for the interpretation of a wide range of existing data and the design of further experiments to elucidate signalling mechanisms within the core signalling unit and larger array

Aqueous dispersion state of amphiphilic hybrid aluminosilicate nanotubes

International audienceAbstract Imogolite is known to form bundles when dried; but these nanotubes can also be perfectly dispersed in water. When the silicon precursor in the synthesis of imogolite nanotubes is changed from tetraethoxysilane (TEOS) to methyltriethoxysilane (TEMS), internal wall hydroxyls are replaced with methyl groups. This chemical modification also happens to change their dispersion properties; the nanotubes then form small bundles in solution. In this work, we explore modifications of the synthesis procedure in order to enhance the dispersion of such hybrid nanotubes. The study focuses on the effects of [Al]/[Si] ratio, acid types, ethanol addition or choice of silicon precursor. Using small-angle X-ray scattering (SAXS), infra-red spectroscopy (IR) and cryo-TEM, we show that it is possible to improve yield and length. However, none of the tested modifications allowed the prevention of bundle formation in solution

Structural landscape of the respiratory syncytial virus nucleocapsids

Human Respiratory Syncytial Virus (HRSV) is a prevalent cause of severe respiratory infections in children and the elderly. The helical HRSV nucleocapsid is a template for the viral RNA synthesis and a scaffold for the virion assembly. This cryo-electron microscopy analysis reveals the non-canonical arrangement of the HRSV nucleocapsid helix, composed of 16 nucleoproteins per asymmetric unit, and the resulting systematic variations in the RNA accessibility. We demonstrate that this unique helical symmetry originates from longitudinal interactions by the C-terminal arm of the HRSV nucleoprotein. We explore the polymorphism of the nucleocapsid-like assemblies, report five structures of the full-length particles and two alternative arrangements formed by a C-terminally truncated nucleoprotein mutant, and demonstrate the functional importance of the identified longitudinal interfaces. We put all these findings in the context of the HRSV RNA synthesis machinery and delineate the structural basis for its further investigation

Measuring Particle Size Distribution by Asymmetric Flow Field Flow Fractionation: A Powerful Method for the Preclinical Characterization of Lipid-Based Nanoparticles

International audienceParticle size distribution and stability are key attributes for the evaluation of the safety and efficacy profile of medical nanoparticles (Med-NPs). Measuring particle average size and particle size distribution is a challenging task which requires the combination of orthogonal high-resolution sizing techniques, especially in complex biological media. Unfortunately, despite its limitations, due to its accessibility, low cost, and easy handling, batch mode dynamic light scattering (DLS) is still very often used as the only approach to measure particle size distribution in the nanomedicine field. In this work the use of asymmetric flow field flow fractionation coupled to multiangle light scattering and dynamic light scattering detectors (AF4-MALS-DLS) was evaluated as an alternative to batch mode DLS to measure the physical properties of lipid-based nanoparticles. A robust standard operating procedure (SOPs) developed by the Nanomedicine Characterization Laboratory (EUNCL) was presented and tested to assess size stability, batch to batch consistency, and the behavior of the lipid-based nanoparticles in plasma. Orthogonal sizing techniques, such as transmission electron microscopy (TEM) and particle tracking analysis (PTA) measurements, were performed to support the results. While batch mode DLS could be applied as a fast and simple method to provide a preliminary insight into the integrity and polydispersity of samples, it was unsuitable to resolve small modifications of the particle size distribution. The introduction of nanoparticle sorting by field-flow fractionation coupled to online DLS and MALS allowed assessment of batch to batch variability and changes in the size of the lipid nanoparticles induced by the interaction with serum proteins, which are critical for quality control and regulatory aspects. In conclusion, if a robust SOP is followed, AF4-MALS-DLS is a powerful method for the preclinical characterization of lipid-based nanoparticles

Assembly principles of a unique cage formed by hexameric and decameric E. coli proteins.

International audienceA 3.3 MDa macromolecular cage between two Escherichia coli proteins with seemingly incompatible symmetries-the hexameric AAA+ ATPase RavA and the decameric inducible lysine decarboxylase LdcI-is reconstructed by cryo-electron microscopy to 11 Å resolution. Combined with a 7.5 Å resolution reconstruction of the minimal complex between LdcI and the LdcI-binding domain of RavA, and the previously solved crystal structures of the individual components, this work enables to build a reliable pseudoatomic model of this unusual architecture and to identify conformational rearrangements and specific elements essential for complex formation. The design of the cage created via lateral interactions between five RavA rings is unique for the diverse AAA+ ATPase superfamily

Hexagonalization of Aluminogermanate Imogolite Nanotubes Organized into Closed-Packed Bundles

Imogolite nanotubes are promising building blocks for nanotechnologies with potential applications in molecular separation, molecular storage, or catalysis. We present an experimental study of the structure of germanium-based imogolite nanotubes Al₂O₃Ge­(OH)₄ arranged in bundles. It combines cryo-transmission electron microscopy, infrared spectroscopy, thermogravimetric measurements, and X-ray scattering experiments. Thanks to a systematic method developed to analyze X-ray scattering diagrams as a function of the nanotube shape, single-walled germanium-based imogolite nanotubes, known as cylindrical for more than 30 years, are shown to take an hexagonal base shape when arranged in bundles. Physical and chemical properties of hexagonal imogolite nanotubes should markedly differ from those of cylindrical ones, making hexagonal basis nanotubes a “new” member, of particular interest, of the rich family of imogolites

Green Nanovectors for Phytodrug Delivery: In-Depth Structural and Morphological Characterization

Lipid-based soft nanovectors are widely used for delivery purposes in many fields of applications. Although it is generally considered a class of molecules with superior biocompatibility, in recent times researchers have expressed concerns with regards to the sustainability of preparation procedures. In particular, when large-scale production is involved, as in the case of agro-nanotechnology applications, this becomes a most pressing issue. Recently, we proposed novel green nanoformulations for the delivery of phytohormones (i.e., indole-3-butyric acid and 1-naphthaleneacetic acid) to Olea europaea L., obtained from agricultural waste derived from the plant itself, which yielded promising results, especially when engineered by addition of pure phospholipids in small amounts (10% w/w). Here we have performed an advanced analysis of high-resolution structural data from X-ray and neutron scattering (SAXS/SANS) and cryo-transmission electron microscopy (cryo-TEM). The results obtained from these combined techniques were complementary and fully consistent, evidencing the fundamental role of the employed adjuvant phospholipid in dictating the structural arrangement

Structural and biochemical characterisation of the Providencia stuartii arginine decarboxylase shows distinct polymerisation and regulation

International audienceAbstract Bacterial homologous lysine and arginine decarboxylases play major roles in the acid stress response, physiology, antibiotic resistance and virulence. The Escherichia coli enzymes are considered as their archetypes. Whereas acid stress triggers polymerisation of the E. coli lysine decarboxylase LdcI, such behaviour has not been observed for the arginine decarboxylase Adc. Here we show that the Adc from a multidrug-resistant human pathogen Providencia stuartii massively polymerises into filaments whose cryo-EM structure reveals pronounced differences between Adc and LdcI assembly mechanisms. While the structural determinants of Adc polymerisation are conserved only in certain Providencia and Burkholderia species , acid stress-induced polymerisation of LdcI appears general for enterobacteria. Analysis of the expression, activity and oligomerisation of the P. stuartii Adc further highlights the distinct properties of this unusual protein and lays a platform for future investigation of the role of supramolecular assembly in the superfamily or arginine and lysine decarboxylases

Nonclassical Growth Mechanism of Double‐Walled Metal‐Oxide Nanotubes Implying Transient Single‐Walled Structures

International audienceThe formation of imogolite nanotubes is reported to be a kinetic process involving intermediate roof‐tile nanostructures. Here, the structural evolution occurring during the synthesis of aluminogermanate double‐walled imogolite nanotubes is in situ monitored, thanks to an instrumented autoclave allowing the control of the temperature, the continuous measurement of pH and pressure, and the regular sampling of gas and solution. Chemical analyses confirm the completion of the precursor's conversion with the release of CO2 , ethanol, and dioxane as main side products. The combination of microscopic observations, infrared, and absorption spectroscopies with small and wide‐angle X‐ray scattering experiments unravel a unique growth mechanism implying transient single‐walled nanotubes instead of the self‐assembly of stacked proto‐imogolite tiles. The growth formation of these transient nanotubes is followed at the molecular level by Quick‐X‐ray absoprtion specotrscopy experiments. Multivariate data analysis evidences that the near neighboring atomic environment of Ge evolves from monotonous to a more complex one as the reaction progresses. The following transformation into a double‐walled nanotube takes place at a nearly constant mean radius, as demonstrated by the simulation of X‐ray scattering diagrams. Overall, transient nanotubes appear to serve for the anchoring of a new wall, corresponding to a mechanism radically different from that proposed in the literature