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.
New methods to automatically build models of macromolecular complexes from high-resolution structures or homology models of their subunits or domains against x-ray or neutron small-angle scattering ...data are presented. Depending on the complexity of the object, different approaches are employed for the global search of the optimum configuration of subunits fitting the experimental data. An exhaustive grid search is used for hetero- and homodimeric particles and for symmetric oligomers formed by identical subunits. For the assemblies or multidomain proteins containing more then one subunit/domain per asymmetric unit, heuristic algorithms based on simulated annealing are used. Fast computational algorithms based on spherical harmonics representation of scattering amplitudes are employed. The methods allow one to construct interconnected models without steric clashes, to account for the particle symmetry and to incorporate information from other methods, on distances between specific residues or nucleotides. For multidomain proteins, addition of missing linkers between the domains is possible. Simultaneous fitting of multiple scattering patterns from subcomplexes or deletion mutants is incorporated. The efficiency of the methods is illustrated by their application to complexes of different types in several simulated and practical examples. Limitations and possible ambiguity of rigid body modeling are discussed and simplified docking criteria are provided to rank multiple models. The methods described are implemented in publicly available computer programs running on major hardware platforms.
Structural analysis of flexible macromolecular systems such as intrinsically disordered or multidomain proteins with flexible linkers is a difficult task as high-resolution techniques are barely ...applicable. A new approach, ensemble optimization method (EOM), is proposed to quantitatively characterize flexible proteins in solution using small-angle X-ray scattering (SAXS). The flexibility is taken into account by allowing for the coexistence of different conformations of the protein contributing to the experimental scattering pattern. These conformers are selected using a genetic algorithm from a pool containing a large number of randomly generated models covering the protein configurational space. Quantitative criteria are developed to analyze the EOM selected models and to determine the optimum number of conformers in the ensemble. Simultaneous fitting of multiple scattering patterns from deletion mutants, if available, provides yet more detailed local information about the structure. The efficiency of EOM is demonstrated in model and practical examples on completely or partially unfolded proteins and on multidomain proteins interconnected by linkers. In the latter case, EOM is able to distinguish between rigid and flexible proteins and to directly assess the interdomain contacts.
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.
New developments in small-angle X-ray and neutron scattering studies of biological macromolecules in solution are presented. Small-angle scattering is rapidly becoming a streamline tool in structural ...molecular biology providing unique information about overall structure and conformational changes of native individual proteins, functional complexes, flexible macromolecules and assembly processes.
Small‐angle scattering (SAS) is frequently employed for screening large numbers of samples and for studying these samples under different conditions, including space‐ and time‐resolved analysis. ...These measurements produce immense amounts of data, especially on modern high‐flux and high‐brilliance sources (e.g. third‐generation synchrotrons). In biological SAS, like high‐throughput macromolecular crystallography, large‐scale analysis of proteins and macromolecular complexes is also emerging. Automation of data analysis becomes an indispensable prerequisite for adequate evaluation of high‐throughput SAS experiments. Here a prototype of an automated data‐analysis system for isotropic solution scattering based on the further development of the programs belonging to the package ATSAS 2.1 is reported. This system allows the major analysis tasks starting from the raw data processing and, for monodisperse systems, finishing with a three‐dimensional model, to be performed automatically. Convenient web interfaces for the online use of individual ATSAS programs are also provided.
Roundabout (Robo) receptors provide an essential repulsive cue in neuronal development following Slit ligand binding. This important signaling pathway can also be hijacked in numerous cancers, making ...Slit-Robo an attractive therapeutic target. However, little is known about how Slit binding mediates Robo activation. Here we present the crystal structure of Robo1 Ig1-4 and Robo1 Ig5, together with a negative stain electron microscopy reconstruction of the Robo1 ectodomain. These results show how the Robo1 ectodomain is arranged as compact dimers, mainly mediated by the central Ig domains, which can further interact in a “back-to-back” fashion to generate a tetrameric assembly. We also observed no change in Robo1 oligomerization upon interaction with the dimeric Slit2-N ligand using fluorescent imaging. Taken together with previous studies we propose that Slit2-N binding results in a conformational change of Robo1 to trigger cell signaling.
Display omitted
•Robo1 Ig1-Ig4 adopts an extended conformation•Robo1 ectodomain forms a compact tetrameric assembly in vitro•No change in Robo1 oligomerization occurs upon interaction with Slit2-N
Slit-Robo signaling is an essential neuronal development pathway, but little is known about how Slit binding to Robo is transmitted across the cell membrane. Aleksandrova et al. present a structural model of a Robo ectodomain to provide insight into receptor activation upon Slit binding.
Display omitted
•Dps protects bacterial genome from harmful effects by co-crystallization with DNA.•DNA does not wrap Dps molecules in DNA–Dps complexes in vitro.•Dps molecule contacts with a DNA ...segment of ~6 nm length.•DNA is condensed by small quasi-crystal Dps arrangements in vitro.•DNA may arrange along the rows of ordered Dps molecules in DNA–Dps co-crystals.
DNA co-crystallization with Dps family proteins is a fundamental mechanism, which preserves DNA in bacteria from harsh conditions. Though many aspects of this phenomenon are well characterized, the spatial organization of DNA in DNA–Dps co-crystals is not completely understood, and existing models need further clarification. To advance in this problem we have utilized atomic force microscopy (AFM) as the main structural tool, and small-angle X-scattering (SAXS) to characterize Dps as a key component of the DNA-protein complex. SAXS analysis in the presence of EDTA indicates a significantly larger radius of gyration for Dps than would be expected for the core of the dodecamer, consistent with the N-terminal regions extending out into solution and being accessible for interaction with DNA. In AFM experiments, both Dps protein molecules and DNA–Dps complexes adsorbed on mica or highly oriented pyrolytic graphite (HOPG) surfaces form densely packed hexagonal structures with a characteristic size of about 9 nm. To shed light on the peculiarities of DNA interaction with Dps molecules, we have characterized individual DNA–Dps complexes. Contour length evaluation has confirmed the non-specific character of Dps binding with DNA and revealed that DNA does not wrap Dps molecules in DNA–Dps complexes. Angle analysis has demonstrated that in DNA–Dps complexes a Dps molecule contacts with a DNA segment of ~6 nm in length. Consideration of DNA condensation upon complex formation with small Dps quasi-crystals indicates that DNA may be arranged along the rows of ordered protein molecules on a Dps sheet.
Small-angle scattering of X-rays (SAXS) is an established method for low-resolution structural characterization of biological macromolecules in solution. Being complementary to the high resolution ...methods (X-ray crystallography and NMR), SAXS is often used in combination with them. The technique provides overall three-dimensional structures using ab initio reconstructions and hybrid modeling, and allows one to quantitatively characterize equilibrium mixtures as well as flexible systems. Recent progress in SAXS instrumentation, most notably, high brilliance synchrotron sources, has paved the way for high throughput automated SAXS studies allowing screening of external conditions (pH, temperature, ligand binding etc.). The modern approaches for SAXS data analysis are presented in this review including rapid characterization of macromolecular solutions in terms of low-resolution shapes, validation of high-resolution models in close-to-native conditions, quaternary structure analysis of complexes and quantitative description of the oligomeric composition in mixtures. Practical aspects of SAXS as a standalone tool and its combinations with other structural, biophysical or bioinformatics methods are reviewed. The capabilities of the technique are illustrated by a selection of recent applications for the studies of biological molecules. Future perspectives on SAXS and its potential impact to structural molecular biology are discussed.
An
ab initio method for building structural models of proteins from x-ray solution scattering data is presented. Simulated annealing is employed to find a chain-compatible spatial distribution of ...dummy residues which fits the experimental scattering pattern up to a resolution of 0.5
nm. The efficiency of the method is illustrated by the
ab initio reconstruction of models of several proteins, with known and unknown crystal structure, from experimental scattering data. The new method substantially improves the resolution and reliability of models derived from scattering data and makes solution scattering a useful technique in large-scale structural characterization of proteins.
PDF
