ATSAS is a comprehensive software suite for the analysis of small-angle scattering data from dilute solutions of biological macromolecules or nanoparticles. It contains applications for primary data processing and assessment, ab initio bead modelling, and model validation, as well as methods for the analysis of flexibility and mixtures. In addition, approaches are supported that utilize information from X-ray crystallography, nuclear magnetic resonance spectroscopy or atomistic homology modelling to construct hybrid models based on the scattering data. This article summarizes the progress made during the 2.5-2.8 ATSAS release series and highlights the latest developments. These include AMBIMETER, an assessment of the reconstruction ambiguity of experimental data; DATCLASS, a multiclass shape classification based on experimental data; SASRES, for estimating the resolution of ab initio model reconstructions; CHROMIXS, a convenient interface to analyse in-line size exclusion chromatography data; SHANUM, to evaluate the useful angular range in measured data; SREFLEX, to refine available high-resolution models using normal mode analysis; SUPALM for a rapid superposition of low-and highresolution models; and SASPy, the ATSAS plugin for interactive modelling in PyMOL. All these features and other improvements are included in the ATSAS release 2.8, freely available for academic users from https://www.emblhamburg.de/biosaxs/software.html.

The development of RP101 as a cancer drug represents a truly novel approach for prevention of chemoresistance and enhancement of chemosensitivity.

The tropoelastin monomer undergoes stages of association by coacervation, deposition onto microfibrils, and cross-linking to form elastic fibers. Tropoelastin consists of an elastic N-terminal coil region and a cell-interactive C-terminal foot region linked together by a highly exposed bridge region. The bridge region is conveniently positioned to modulate elastic fiber assembly through association by coacervation and its proximity to dominant cross-linking domains. Tropoelastin constructs that either modify or remove the entire bridge and downstream regions were assessed for elastogenesis. These constructs focused on a single alanine substitution (R515A) and a truncation (M155n) at the highly conserved arginine 515 site that borders the bridge. Each form displayed less efficient coacervation, impaired hydrogel formation, and decreased dermal fibroblast attachment compared to wild-type tropoelastin. The R515A mutant protein additionally showed reduced elastic fiber formation upon addition to human retinal pigmented epithelium cells and dermal fibroblasts. The small-angle X-ray scattering nanostructure of the R515A mutant protein revealed greater conformational flexibility around the bridge and C-terminal regions. This increased flexibility of the R515A mutant suggests that the tropoelastin R515 residue stabilizes the structure of the bridge region, which is critical for elastic fiber assembly.tropoelastin assembly | protease resistance E lastic fibers confer the elastic and recoil properties required for repetitive and reversible deformation of elastic tissues during normal function (1-3). The assembly of elastic fibers from tropoelastin, the soluble precursor of elastin, is classically defined by stages of tropoelastin synthesis, coacervation, microfibrillar deposition, and cross-linking. Tropoelastin is secreted by elastogenic cells such as smooth muscle cells, endothelial cells, and fibroblasts (1, 2). At the cell surface, the monomers cluster through hydrophobic domain interactions in an aqueous environment (3-6) by the entropically driven process of coacervation (7). These tropoelastin assemblies remain attached through the C terminus to cell-surface integrin αvβ3 and glycosaminoglycans (8-10) until deposition on microfibrillar scaffolds (11), which direct the shape and orientation of elastic fibers (10, 12, 13). Microfibrillar proteins recruit lysyl oxidase (14, 15), which reacts with specific tropoelastin lysine residues to form cross-links (1, 11, 16). These cross-links occur at multiple sites in the molecule (17) and are enriched in domains 19-25 (1, 16). Cross-linking imposes expansional constraints on elastin and renders elastic fibers resilient under repetitive mechanical stretching (1,12,18).The recently solved nanostructure of tropoelastin has allowed the main functional regions of tropoelastin to be placed within a structural context (19). Most of the elasticity of the molecule is conferred by a coiled region (20) that extends from domain 2 to domain 18. A protrusion from the coil region around do...

Summary Toxin-antitoxin (TA) systems regulate fundamental cellular processes in bacteria and represent potential therapeutic targets. We report a new RES-Xre TA system in multiple human pathogens, including Mycobacterium tuberculosis. The toxin, MbcT, is bactericidal unless neutralized by its antitoxin MbcA. To investigate the mechanism, we solved the 1.8 Å-resolution crystal structure of the MbcTA complex. We found that MbcT resembles secreted NAD + -dependent bacterial exotoxins, such as diphtheria toxin. Indeed, MbcT catalyzes NAD + degradation in vitro and in vivo . Unexpectedly, the reaction is stimulated by inorganic phosphate, and our data reveal that MbcT is a NAD + phosphorylase. In the absence of MbcA, MbcT triggers rapid M. tuberculosis cell death, which reduces mycobacterial survival in macrophages and prolongs the survival of infected mice. Our study expands the molecular activities employed by bacterial TA modules and uncovers a new class of enzymes that could be exploited to treat tuberculosis and other infectious diseases.

GlgE (EC 2.4.99.16) is an α-maltose 1-phosphate:(1→4)-α-d-glucan 4-α-d-maltosyltransferase of the CAZy glycoside hydrolase 13_3 family. It is the defining enzyme of a bacterial α-glucan biosynthetic pathway and is a genetically validated anti-tuberculosis target. It catalyzes the α-retaining transfer of maltosyl units from α-maltose 1-phosphate to maltooligosaccharides and is predicted to use a double-displacement mechanism. Evidence of this mechanism was obtained using a combination of site-directed mutagenesis of Streptomyces coelicolor GlgE isoform I, substrate analogues, protein crystallography, and mass spectrometry. The X-ray structures of α-maltose 1-phosphate bound to a D394A mutein and a β-2-deoxy-2-fluoromaltosyl-enzyme intermediate with a E423A mutein were determined. There are few examples of CAZy glycoside hydrolase family 13 members that have had their glycosyl-enzyme intermediate structures determined, and none before now have been obtained with a 2-deoxy-2-fluoro substrate analogue. The covalent modification of Asp394 was confirmed using mass spectrometry. A similar modification of wild-type GlgE proteins from S. coelicolor and Mycobacterium tuberculosis was also observed. Small-angle X-ray scattering of the M. tuberculosis enzyme revealed a homodimeric assembly similar to that of the S. coelicolor enzyme but with slightly differently oriented monomers. The deeper understanding of the structure–function relationships of S. coelicolor GlgE will aid the development of inhibitors of the M. tuberculosis enzyme.

A quantitative resolution measure of the ab initio shapes restored from small-angle scattering data is introduced based on the variability of multiple reconstructions. The new measure is validated in simulated examples and its efficiency has been demonstrated in applications to experimental data.

Long-chain bacterial polysaccharides play important roles in pathogenicity. In Escherichia coli O9a, a model for ABC transporter dependent polysaccharide assembly, a large extracellular carbohydrate with a narrow distribution of size is polymerized from monosaccharides by a complex of two proteins, WbdA (polymerase) and WbdD (terminating protein). Such careful control of polymerization is recurring theme in biology. Combining crystallography and small angle X-ray scattering, we show that the C-terminal domain of WbdD contains an extended coiled-coil that physically separates WbdA from the catalytic domain of WbdD. The effects of insertions and deletions within the coiled-coil region were analyzed in vivo, revealing that polymer size is controlled by varying the length of the coiled-coil domain. Thus, the coiled-coil domain of WbdD functions as a molecular ruler that, along with WbdA:WbdD stoichiometry, controls the chain length of a model bacterial polysaccharide.

Many medically relevant Gram-negative bacteria use the type III secretion system (T3SS) to translocate effector proteins into the host for their invasion and intracellular survival. A multiprotein complex located at the cytosolic interface of the T3SS is proposed to act as a sorting platform by selecting and targeting substrates for secretion through the system. However, the precise stoichiometry and 3D organization of the sorting platform components is unknown. Here we reconstitute soluble complexes of the Salmonella Typhimurium sorting platform proteins including the ATPase InvC, the regulator OrgB, the protein SpaO and a recently identified subunit SpaO C, which we show to be essential for the solubility of SpaO. We establish domaindomain interactions, determine for the first time the stoichiometry of each subunit within the complexes by native mass spectrometry and gain insight into their organization using small-angle X-ray scattering. Importantly, we find that in solution the assembly of SpaO/SpaO C /OrgB/InvC adopts an extended L-shaped conformation resembling the sorting platform pods seen in in situ cryo-electron tomography, proposing that this complex is the core building block that can be conceivably assembled into higher oligomers to form the T3SS sorting platform. The determined molecular arrangements of the soluble complexes of the sorting platform provide important insights into its architecture and assembly.