The ATSAS software suite encompasses a number of programs for the processing, visualization, analysis and modelling of small‐angle scattering data, with a focus on the data measured from biological ...macromolecules. Here, new developments in the ATSAS 3.0 package are described. They include IMSIM, for simulating isotropic 2D scattering patterns; IMOP, to perform operations on 2D images and masks; DATRESAMPLE, a method for variance estimation of structural invariants through parametric resampling; DATFT, which computes the pair distance distribution function by a direct Fourier transform of the scattering data; PDDFFIT, to compute the scattering data from a pair distance distribution function, allowing comparison with the experimental data; a new module in DATMW for Bayesian consensus‐based concentration‐independent molecular weight estimation; DATMIF, an ab initio shape analysis method that optimizes the search model directly against the scattering data; DAMEMB, an application to set up the initial search volume for multiphase modelling of membrane proteins; ELLLIP, to perform quasi‐atomistic modelling of liposomes with elliptical shapes; NMATOR, which models conformational changes in nucleic acid structures through normal mode analysis in torsion angle space; DAMMIX, which reconstructs the shape of an unknown intermediate in an evolving system; and LIPMIX and BILMIX, for modelling multilamellar and asymmetric lipid vesicles, respectively. In addition, technical updates were deployed to facilitate maintainability of the package, which include porting the PRIMUS graphical interface to Qt5, updating SASpy – a PyMOL plugin to run a subset of ATSAS tools – to be both Python 2 and 3 compatible, and adding utilities to facilitate mmCIF compatibility in future ATSAS releases. All these features are implemented in ATSAS 3.0, freely available for academic users at https://www.embl‐hamburg.de/biosaxs/software.html.
ATSAS is a comprehensive software suite for the processing, visualization, analysis and modelling of small‐angle scattering data. This article describes developments in the ATSAS 3.0 release, including new programs for data simulation and for the structural modelling of lipids, nucleic acids and polydisperse systems.
Small‐angle X‐ray scattering (SAXS) is an established technique for structural analysis of biological macromolecules in solution. During the last decade, inline chromatography setups coupling SAXS ...with size exclusion (SEC‐SAXS) or ion exchange (IEC‐SAXS) have become popular in the community. These setups allow one to separate individual components in the sample and to record SAXS data from isolated fractions, which is extremely important for subsequent data interpretation, analysis, and structural modeling. However, in case of partially overlapping elution peaks, inline chromatography SAXS may still yield scattering profiles from mixtures of components. The deconvolution of these scattering data into the individual fractions is nontrivial and potentially ambiguous. We describe a cross‐platform computer program, EFAMIX, for restoring the scattering and concentration profiles of the components based on the evolving factor analysis (EFA). The efficiency of the program is demonstrated in a number of simulated and experimental SEC‐SAXS data sets. Sensitivity and limitations of the method are explored, and its applicability to IEC‐SAXS data is discussed. EFAMIX requires minimal user intervention and is available to academic users through the program package ATSAS as from release 3.1.
New developments in the program package ATSAS (version 2.4) for the processing and analysis of isotropic small‐angle X‐ray and neutron scattering data are described. They include (i) multiplatform ...data manipulation and display tools, (ii) programs for automated data processing and calculation of overall parameters, (iii) improved usage of high‐ and low‐resolution models from other structural methods, (iv) new algorithms to build three‐dimensional models from weakly interacting oligomeric systems and complexes, and (v) enhanced tools to analyse data from mixtures and flexible systems. The new ATSAS release includes installers for current major platforms (Windows, Linux and Mac OSX) and provides improved indexed user documentation. The web‐related developments, including a user discussion forum and a widened online access to run ATSAS programs, are also presented.
The spectrin superfamily of proteins plays key roles in assembling the actin cytoskeleton in various cell types, crosslinks actin filaments, and acts as scaffolds for the assembly of large protein ...complexes involved in structural integrity and mechanosensation, as well as cell signaling. α-actinins in particular are the major actin crosslinkers in muscle Z-disks, focal adhesions, and actin stress fibers. We report a complete high-resolution structure of the 200 kDa α-actinin-2 dimer from striated muscle and explore its functional implications on the biochemical and cellular level. The structure provides insight into the phosphoinositide-based mechanism controlling its interaction with sarcomeric proteins such as titin, lays a foundation for studying the impact of pathogenic mutations at molecular resolution, and is likely to be broadly relevant for the regulation of spectrin-like proteins.
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•Structure of human α-actinin-2 in an autoinhibited closed conformation•Facilitation of PIP2-induced allosteric modulation for opening and titin binding•Essentiality of structural flexibility for crosslinking antiparallel F-actin•Relevance for the intramolecular pseudoligand regulation mechanism of the spectrin family
The structure of human α-actinin, the major component of the basic contractile units of muscles, reveals a unique phosphoinositide-based mechanism of its regulation, as well as assembly.
A critical quality attribute for liquid formulations is the absence of visible particles. Such particles may form upon polysorbate hydrolysis resulting in release of free fatty acids into solution ...followed by precipitation. Strategies to avoid this effect are of major interest for the pharmaceutical industry. In this context, we investigated the structural organization of polysorbate micelles alone and upon addition of the fatty acid myristic acid (MA) by small-angle x-ray scattering. Two complementary approaches using a model of polydisperse core-shell ellipsoidal micelles and an ensemble of quasiatomistic micelle structures gave consistent results well describing the experimental data. The small-angle x-ray scattering data reveal polydisperse mixtures of ellipsoidal micelles containing about 22–35 molecules per micelle. The addition of MA at concentrations up to 100 μg/mL reveals only marginal effects on the scattering data. At the same time, addition of high amounts of MA (>500 μg/mL) increases the average sizes of the micelles indicating that MA penetrates into the surfactant micelles. These results together with molecular modeling shed light on the polysorbate contribution to fatty acid solubilization preventing or delaying fatty acid particle formation.
Ataxin-3 is a deubiquitinase involved in protein quality control and other essential cellular functions. It preferentially interacts with polyubiquitin chains of four or more units attached to ...proteins delivered to the ubiquitin-proteasome system. Ataxin-3 is composed of an N-terminal Josephin domain and a flexible C terminus that contains two or three ubiquitin-interacting motifs (UIMs) and a polyglutamine tract, which, when expanded beyond a threshold, leads to protein aggregation and misfolding and causes spinocerebellar ataxia type 3. The high-resolution structure of the Josephin domain is available, but the structural and dynamical heterogeneity of ataxin-3 has so far hindered the structural description of the full-length protein. Here, we characterize non-expanded and expanded variants of ataxin-3 in terms of conformational ensembles adopted by the proteins in solution by jointly using experimental data from nuclear magnetic resonance and small-angle X-ray scattering with coarse-grained simulations. Our results pave the way to a molecular understanding of polyubiquitin recognition.
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•Expansion of a polyglutamine tract in ataxin-3 leads to spinocerebellar ataxia type 3•We characterized the conformational ensemble of expanded and non-expanded ataxin-3•We demonstrate that the C terminus samples sparsely populated collapsed conformations•Conformational plasticity of ataxin-3 may play a role in normal function and disease
Using integrative structural biology methods, Sicorello et al. characterized the conformation of full-length ataxin-3, a deubiquitinase containing an N-terminal globular domain and a C-terminal intrinsically disordered region. The results contribute to our understanding of the role that the conformational plasticity of ataxin-3 plays in normal function and in disease.
Small‐angle X‐ray scattering (SAXS) is widely utilized to study soluble macromolecules, including those embedded into lipid carriers and delivery systems such as surfactant micelles, phospholipid ...vesicles and bilayered nanodiscs. To adequately describe the scattering from such systems, one needs to account for both the form factor (overall structure) and long‐range‐order Bragg reflections emerging from the organization of bilayers, which is a non‐trivial task. Presently existing methods separate the analysis of lipid mixtures into distinct procedures using form‐factor fitting and the fitting of the Bragg peak regions. This article describes a general approach for the computation and analysis of SAXS data from lipid mixtures over the entire angular range of an experiment. The approach allows one to restore the electron density of a lipid bilayer and simultaneously recover the corresponding size distribution and multilamellar organization of the vesicles. The method is implemented in a computer program, LIPMIX, and its performance is demonstrated on an aqueous solution of layered lipid vesicles undergoing an extrusion process. The approach is expected to be useful for the analysis of various types of lipid‐based systems, e.g. for the characterization of interactions between target drug molecules and potential carrier/delivery systems.
An approach to restore the structural parameters of lipid mixtures from small‐angle X‐ray scattering data is developed.
The program package ATSAS 2.1 for small‐angle X‐ray and neutron scattering data analysis is presented. The programs included in the package cover the major processing and interpretation steps from ...primary data reduction to three‐dimensional modelling. This system is primarily oriented towards the analysis of biological macromolecules, but could also be used for non‐biological isotropic and partially ordered objects (nanoparticle systems, colloidal solutions, polymers in solution and bulk). Recent developments in the programs included in ATSAS 2.1 are highlighted. The main programs run on multiple hardware platforms, including Windows PC, Linux RedHat and Suse, DEC Alpha, SGI IRIX and Mac OSX.
Emfourin (M4in) is a protein metalloprotease inhibitor recently discovered in the bacterium Serratia proteamaculans and the prototype of a new family of protein protease inhibitors with an unknown ...mechanism of action. Protealysin-like proteases (PLPs) of the thermolysin family are natural targets of emfourin-like inhibitors widespread in bacteria and known in archaea. The available data indicate the involvement of PLPs in interbacterial interaction as well as bacterial interaction with other organisms and likely in pathogenesis. Arguably, emfourin-like inhibitors participate in the regulation of bacterial pathogenesis by controlling PLP activity. Here, we determined the 3D structure of M4in using solution NMR spectroscopy. The obtained structure demonstrated no significant similarity to known protein structures. This structure was used to model the M4in–enzyme complex and the complex model was verified by small-angle X-ray scattering. Based on the model analysis, we propose a molecular mechanism for the inhibitor, which was confirmed by site-directed mutagenesis. We show that two spatially close flexible loop regions are critical for the inhibitor–protease interaction. One region includes aspartic acid forming a coordination bond with catalytic Zn2+ of the enzyme and the second region carries hydrophobic amino acids interacting with protease substrate binding sites. Such an active site structure corresponds to the noncanonical inhibition mechanism. This is the first demonstration of such a mechanism for protein inhibitors of thermolysin family metalloproteases, which puts forward M4in as a new basis for the development of antibacterial agents relying on selective inhibition of prominent factors of bacterial pathogenesis belonging to this family.
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