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Transient biomolecular interactions play crucial roles in many cellular signaling and regulation processes. However, deciphering the structure of these assemblies is challenging owing to the difficulties in isolating complexes from the individual partners. The additive nature of small-angle X-ray scattering (SAXS) data allows for probing the species present in these mixtures, but decomposition into structural and thermodynamic information is difficult. We present a chemometric approach enabling the decomposition of titration SAXS data into species-specific information. Using extensive synthetic SAXS data, we demonstrate that robust decomposition can be achieved for titrations with a maximum fraction of complex of 0.5 that can be extended to 0.3 when two orthogonal titrations are simultaneously analyzed. The effect of the structural features, titration points, relative concentrations, and noise are thoroughly analyzed. The validation of the strategy with experimental data highlights the power of the approach to provide unique insights into this family of biomolecular assemblies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Small-angle X-ray scattering (SAXS) is a low-resolution method for the structural characterization of biological macromolecules in solution. Information about the overall structural features provided by SAXS is highly complementary to X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy, which are high-resolution methods. SAXS not only provides the shape, oligomeric state, and quaternary structure of folded proteins and protein complexes but also allows for quantitative analysis of flexible biomolecules. In this chapter, the most relevant SAXS procedures for structural characterization of flexible macromolecules, including intrinsically disordered proteins (IDPs), are presented. The sample requirements for SAXS experiments on protein solutions and the sequence of steps in data collection and processing are described. The use of the advanced data analysis tools to quantitatively characterize flexible proteins is presented in detail. Typical experimental issues and potential problems encountered during SAXS data measurements and analyses are discussed.

Clarification of solution structure and its modulation in proteins and protein complexes is crucially important to understand dynamical ordering in macromolecular systems. Small-angle x-ray scattering (SAXS) and small-angle neutron scattering (SANS) are among the most powerful techniques to derive structural information. Recent progress in sample preparation, instruments and software analysis is opening up a new era for small-angle scattering. In this review, recent progress and trends of SAXS and SANS are introduced from the point of view of instrumentation and analysis, touching on general features and standard methods of small-angle scattering. This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato., (Copyright © 2017. Published by Elsevier B.V.)

Intrinsically Disordered Proteins (IDPs) perform a broad range of biological functions. Their relevance has motivated intense research activity seeking to characterize their sequence/structure/function relationships. However, the conformational plasticity of these molecules hampers the application of traditional structural approaches, and new tools and concepts are being developed to address the challenges they pose. Small-Angle Scattering (SAS) is a structural biology technique that probes the size and shape of disordered proteins and their complexes with other biomolecules. The low-resolution nature of SAS can be compensated with specially designed computational tools and its combined interpretation with complementary structural information. In this review, we describe recent advances in the application of SAS to disordered proteins and highly flexible complexes and discuss current challenges., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Formation of amyloids is the hallmark of several neurodegenerative pathologies. Structural investigation of these complex transformation processes poses significant experimental challenges due to the co-existence of multiple species. The additive nature of small-angle X-ray scattering (SAXS) data allows for probing the evolution of these mixtures of oligomeric states, but the decomposition of SAXS data into species-specific spectra and relative concentrations is burdened by ambiguity. We present an objective SAXS data decomposition method by adapting the multivariate curve resolution alternating least squares (MCR-ALS) chemometric method. The approach enables rigorous and robust decomposition of synchrotron SAXS data by simultaneously introducing these data in different representations that emphasize molecular changes at different time and structural resolution ranges. The approach has allowed the study of fibrillogenic forms of insulin and the familial mutant E46K of α-synuclein, and is generally applicable to any macromolecular mixture that can be probed by SAXS., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Intrinsically Disordered Proteins (IDPs) are fundamental actors of biological processes. Their inherent plasticity facilitates very specialized tasks in cell regulation and signalling, and their malfunction is linked to severe pathologies. Understanding the functional role of disorder requires the structural characterization of IDPs and the complexes they form. Small-angle Scattering of X-rays (SAXS) and Neutrons (SANS) have notably contributed to this structural understanding. In this review we summarize the most relevant developments in the field of SAS studies of disordered proteins. Emphasis is given to ensemble methods and how SAS data can be combined with computational approaches or other biophysical information such as NMR. The unique capabilities of SAS enable its application to extremely challenging disordered systems such as low-complexity regions, amyloidogenic proteins and transient biomolecular complexes. This reinforces the fundamental role of SAS in the structural and dynamic characterization of this elusive family of proteins.

Structural characterization of protein-protein interactions at molecular level is essential to understand biological processes and identify new therapeutic opportunities. However, atomic resolution structural techniques cannot keep pace with current advances in interactomics. Low-resolution structural techniques, such as small-angle X-ray scattering (SAXS), can be applied at larger scale, but they miss atomic details. For efficient application to protein-protein complexes, low-resolution information can be combined with theoretical methods that provide energetic description and atomic details of the interactions. Here we present the pyDockSAXS web server (http://life.bsc.es/pid/pydocksaxs) that provides an automatic pipeline for modeling the structure of a protein-protein complex from SAXS data. The method uses FTDOCK to generate rigid-body docking models that are subsequently evaluated by a combination of pyDock energy-based scoring function and their capacity to describe SAXS data. The only required input files are structural models for the interacting partners and a SAXS curve. The server automatically provides a series of structural models for the complex, sorted by the pyDockSAXS scoring function. The user can also upload a previously computed set of docking poses, which opens the possibility to filter the docking solutions by potential interface residues or symmetry restraints. The server is freely available to all users without restriction., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Small-angle scattering of X-rays (SAXS) is a method for the low-resolution structural characterization of biological macromolecules in solution. The technique is highly complementary to the high-resolution methods of X-ray crystallography and NMR. SAXS not only provides shapes, oligomeric state, and quaternary structures of folded proteins and protein complexes but also allows for the quantitative analysis of flexible systems. Here, major procedures are presented to characterize intrinsically disordered proteins (IDPs) using SAXS. The sample requirements for SAXS experiments on protein solutions are given and the sequence of steps in data collection and processing is described. The use of the recently developed advanced computational tools to quantitatively characterize solutions of IDPs is presented in detail. Typical experimental and potential problems encountered during the use of SAXS are discussed.

Small-angle scattering of X-rays (SAXS) is an established method to study the overall structure and structural transitions of biological macromolecules in solution. For folded proteins, the technique provides three-dimensional low resolution structures ab initio or it can be used to drive rigid-body modeling. SAXS is also a powerful tool for the quantitative analysis of flexible systems, including intrinsically disordered proteins (IDPs), and is highly complementary to the high resolution methods of X-ray crystallography and NMR. Here we present the basic principles of SAXS and review the main approaches to the characterization of IDPs and flexible multidomain proteins using SAXS. Together with the standard approaches based on the analysis of overall parameters, a recently developed Ensemble Optimization Method (EOM) is now available. The latter method allows for the co-existence of multiple protein conformations in solution compatible with the scattering data. Analysis of the selected ensembles provides quantitative information about flexibility and also offers insights into structural features. Examples of the use of SAXS and combined approaches with NMR, X-ray crystallography, and computational methods to characterize completely or partially disordered proteins are presented.

Ribosomal protein L12 is a two-domain protein that forms dimers mediated by its N-terminal domains. A 20-residue linker separates the N- and C-terminal domains. This linker results in a three-lobe topology with significant flexibility, known to be critical for efficient translation. Here we present an ensemble model of spatial distributions and correlation times for the domain reorientations of L12 that reconciles experimental data from small-angle x-ray scattering and nuclear magnetic resonance. We generated an ensemble of L12 conformations in which the structure of each domain is fixed but the domain orientations are variable. The ensemble reproduces the small-angle x-ray scattering data and the optimized correlation times of its reorientational eigenmodes fit the (15)N relaxation data. The ensemble model reveals intrinsic conformational properties of L12 that help explain its function on the ribosome. The two C-terminal domains sample a large volume and extend further away from the ribosome anchor than expected for a random-chain linker, indicating that the flexible linker has residual order. Furthermore, the distances between each C-terminal domain and the anchor are anticorrelated, indicating that one of them is more retracted on average. We speculate that these properties promote the function of L12 to recruit translation factors and control their activity on the ribosome., (Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Multidomain proteins in which consecutive globular regions are connected by linkers are prevalent in nature (Levitt in Proc Natl Acad Sci USA 106:11079-11084, 2009). Some members of this family have largely resisted structural characterization as a result of challenges associated with their inherent flexibility. Small-angle scattering (SAS) is often the method of choice for their structural study. An extensive set of simulated data for both flexible and rigid multidomain systems was analyzed and modeled using standard protocols. This study clearly shows that SAXS profiles obtained from highly flexible proteins can be wrongly interpreted as arising from a rigid structure. In this context, it would be important to identify features from the SAXS data or from the derived structural models that indicate interdomain motions to differentiate between these two scenarios. Features of SAXS data that identify flexible proteins are: (1) general attenuation of fine structure in the scattering profiles, which becomes more dramatic in Kratky representations, and (2) a reduced number of interdomain correlation peaks in p(r) functions that also present large D (max) values and a smooth decrease to 0. When modeling this dynamically averaged SAXS data, the structures obtained present characteristic trends: (1) ab initio models display a decrease in resolution, and (2) rigid-body models present highly extended conformations with a lack of interdomain contacts. The ensemble optimization method represents an excellent strategy to identify interdomain motions unambiguously. This study provides information that should help researchers to select the best modeling strategy for the structural interpretation of SAS experiments of multidomain proteins.

Src kinase plays an important role in several signaling and regulation mechanisms in vivo. Enzymatic activity is tightly regulated through the phosphorylation and dephosphorylation of tyrosine 527, which is placed at the C-terminal tail. Here, we have addressed domain rearrangements involved in the regulation mechanism of Src kinase in solution using small-angle X-ray scattering. In the phosphorylated wild-type form of Src kinase corresponding to the inactive state of the protein, a single conformation compatible with a closed crystallographic structure was found in solution. In the Y527F point mutant representing the active state, analysis of scattering data reveals an equilibrium between two differently populated conformations differing in the radius of gyration by 5 A. The major species (85% of the total population) presents a closed conformation indistinguishable from the crystallographic structure of the inactive state. The minor species (15% of the total population) is an open conformation similar to the crystallographic structure in the active state. The latter structure has the SH3, SH2, and SH2-catalytic domain linker assembled as a pseudo-two-domain protein. The regulation model emerging from this study, including at least three different conformational states, allows the tight regulation of the enzyme without compromising fast response in the presence of natural targets.