In vivo analysis of the Escherichia coli ultrastructure by small-angle scattering

The flagellated Gram-negative bacterium Escherichia coli is one of the most studied microorganisms. Despite extensive studies as a model prokaryotic cell, the ultrastructure of the cell envelope at the nanometre scale has not been fully elucidated. Here, a detailed structural analysis of the bacterium using a combination of small-angle X-ray and neutron scattering (SAXS and SANS, respectively) and ultra-SAXS (USAXS) methods is presented. A multiscale structural model has been derived by incorporating well established concepts in soft-matter science such as a core-shell colloid for the cell body, a multilayer membrane for the cell wall and self-avoiding polymer chains for the flagella. The structure of the cell envelope was resolved by constraining the model by five different contrasts from SAXS, and SANS at three contrast match points and full contrast. This allowed the determination of the membrane electron-density profile and the inter-membrane distances on a quantitative scale. The combination of USAXS and SAXS covers size scales from micrometres down to nanometres, enabling the structural elucidation of cells from the overall geometry down to organelles, thereby providing a powerful method for a noninvasive investigation of the ultrastructure. This approach may be applied for probing in vivo the effect of detergents, antibiotics and antimicrobial peptides on the bacterial cell wall

Macromolecular structure phasing by neutron anomalous diffraction.

In this report we show for the first time that neutron anomalous dispersion can be used in a practical manner to determine experimental phases of a protein crystal structure, providing a new tool for structural biologists. The approach is demonstrated through the use of a state-of-the-art monochromatic neutron diffractometer at the Institut Laue-Langevin (ILL) in combination with crystals of perdeuterated protein that minimise the level of hydrogen incoherent scattering and enhance the visibility of the anomalous signal. The protein used was rubredoxin in which cadmium replaced the iron at the iron-sulphur site. While this study was carried out using a steady-state neutron beam source, the results will be of major interest for capabilities at existing and emerging spallation neutron sources where time-of-flight instruments provide inherent energy discrimination. In particular this capability may be expected to offer unique opportunities to a rapidly developing structural biology community where there is increasing interest in the identification of protonation states, protein/water interactions and protein-ligand interactions - all of which are of central importance to a wide range of fundamental and applied areas in the biosciences

Matchout deuterium labelling of proteins for small-angle neutron scattering studies using prokaryotic and eukaryotic expression systems and high cell-density cultures.

Small-angle neutron scattering (SANS) is a powerful technique for the characterisation of macromolecular structures and interactions. Its main advantage over other solution state approaches is the ability to use D2O/H2O solvent contrast variation to selectively match out specific parts of a multi-component system. While proteins, nucleic acids, and lipids are readily distinguished in this way, it is not possible to locate different parts of a protein-protein system without the introduction of additional contrast by selective deuteration. Here, we describe new methods by which 'matchout labelled' proteins can be produced using Escherichia coli and Pichia pastoris expression systems in high cell-density cultures. The method is designed to produce protein that has a scattering length density that is very close to that of 100% D2O, providing clear contrast when used with hydrogenated partner proteins in a complex. This allows the production of a single sample system for which SANS measurements at different solvent contrasts can be used to distinguish and model the hydrogenated component, the deuterated component, and the whole complex. The approach, which has significant cost advantages, has been extensively tested for both types of expression system

Neutron crystallography reveals mechanisms used by Pseudomonas aeruginosa for host-cell binding

Pseudomonas aeruginosa employs lectins to bind to its host cells, and is known to be the major cause of lung infections. Lectin B (LecB) from Pseudomonas aeruginosa binds specifically to galactose and fucose and is important for pathogenicity, adhesion and biofilm formation. In this work, the neutron crystal structure (1.9 angstrom) of the deuterated LecB/Ca/fucose complex is reported. The structure, in combination with perdeuteration of the ligand and the receptor allowed the observation of hydrogen atoms, protonation states and hydrogen bonds involved in the interaction between pathogenic bacteria and host cells. Thus the study provides structural insights into the mechanism of high affinity binding of LecB to its targets. The opportunistic pathogen Pseudomonas aeruginosa, a major cause of nosocomial infections, uses carbohydrate-binding proteins (lectins) as part of its binding to host cells. The fucose-binding lectin, LecB, displays a unique carbohydrate-binding site that incorporates two closely located calcium ions bridging between the ligand and protein, providing specificity and unusually high affinity. Here, we investigate the mechanisms involved in binding based on neutron crystallography studies of a fully deuterated LecB/fucose/calcium complex. The neutron structure, which includes the positions of all the hydrogen atoms, reveals that the high affinity of binding may be related to the occurrence of a low-barrier hydrogen bond induced by the proximity of the two calcium ions, the presence of coordination rings between the sugar, calcium and LecB, and the dynamic behaviour of bridging water molecules at room temperature. These key structural details may assist in the design of anti-adhesive compounds to combat multi-resistance bacterial infections