Abstract
Summary
Size-exclusion chromatography (SEC) coupled to small-angle X-ray scattering (SAXS), also known as inline SEC-SAXS, is being increasingly used for the structural analysis of ...biological macromolecules, complexes and mixtures in solution. A single SEC-SAXS run generates thousands of individual SAXS profiles from the eluting solute and their analysis requires a correct identification of buffer and sample regions, a rather laborous task. We present CHROMIXS (as in CHROMatography Inline X-ray Scattering), a program for rapid reduction and analysis, both automatically and interactively, of SEC-SAXS data.
Availability and implementation
CHROMIXS is freely available to academic users as part of the ATSAS software suite (www.embl-hamburg.de/biosaxs/download.html).
Small-angle X-ray scattering (SAXS) is a biophysical method to study the overall shape and structural transitions of biological macromolecules in solution. SAXS provides low resolution information on ...the shape, conformation and assembly state of proteins, nucleic acids and various macromolecular complexes. The technique also offers powerful means for the quantitative analysis of flexible systems, including intrinsically disordered proteins (IDPs). Here, the basic principles of SAXS are presented, and profits and pitfalls of the characterization of multidomain flexible proteins and IDPs using SAXS are discussed from the practical point of view. Examples of the synergistic use of SAXS with high resolution methods like X-ray crystallography and nuclear magnetic resonance (NMR), as well as other experimental and in silico techniques to characterize completely, or partially unstructured proteins, are presented.
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.
Structural flexibility and conformational rearrangements are often related to important functions of biological macromolecules, but the experimental characterization of such transitions with ...high-resolution techniques is challenging. At a lower resolution, small angle X-ray scattering (SAXS) can be used to obtain information on biomolecular shapes and transitions in solution. Here, we present SREFLEX, a hybrid modeling approach that uses normal mode analysis (NMA) to explore the conformational space of high-resolution models and refine the structure guided by the agreement with the experimental SAXS data. The method starts from a given conformation of the protein (which does not agree with the SAXS data). The structure is partitioned into pseudo-domains either using structural classification databases or automatically from the protein dynamics as predicted by the NMA. The algorithm proceeds hierarchically employing NMA to first probe large rearrangements and progresses into smaller and more localized movements. At the large rearrangements stage the pseudo-domains stay as rigid bodies allowing one to avoid structural disruptions inherent to the earlier NMA-based algorithms. To validate the approach, we compiled a representative benchmark set of 88 conformational states known experimentally at high resolution. The performance of the algorithm is demonstrated in the simulated data on the benchmark set and also in a number of experimental examples. SREFLEX is included into the ATSAS program package freely available to the academic users, both for download and in the on-line mode.
SREFLEX employs normal mode analysis for the flexible refinement of atomic models of biological macromolecules against solution scattering data, providing insight into conformational transitions.
Small-angle X-ray scattering (SAXS) offers a way to examine the overall shape and oligomerization state of biological macromolecules under quasi native conditions in solution. In the past decades, ...SAXS has become a standard tool for structure biologists due to the availability of high brilliance X-ray sources and the development of data analysis/interpretation methods. Sample handling robots and software pipelines have significantly reduced the time necessary to conduct SAXS experiments. Presently, most synchrotrons feature beamlines dedicated to biological SAXS, and the SAXS-derived models are deposited into dedicated and accessible databases. The size of macromolecules that may be analyzed ranges from small peptides or snippets of nucleic acids to gigadalton large complexes or even entire viruses. Compared to other structural methods, sample preparation is straightforward, and the risk of inducing preparation artefacts is minimal. Very importantly, SAXS is a method of choice to study flexible systems like unfolded or disordered proteins, providing the structural ensembles compatible with the data. Although it may be utilized stand-alone, SAXS profits a lot from available experimental or predicted high-resolution data and information from complementary biophysical methods. Here, we show the basic principles of SAXS and review latest developments in the fields of hybrid modeling and flexible systems.
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•High-throughput screening can be performed at automated synchrotron beamlines.•Hybrid modeling combines SAXS data with high-resolution information.•SAXS allows one to structurally characterize flexible macromolecules.
DAMMIF, a revised implementation of the ab‐initio shape‐determination program DAMMIN for small‐angle scattering data, is presented. The program was fully rewritten, and its algorithm was optimized ...for speed of execution and modified to avoid limitations due to the finite search volume. Symmetry and anisometry constraints can be imposed on the particle shape, similar to DAMMIN. In equivalent conditions, DAMMIF is 25–40 times faster than DAMMIN on a single CPU. The possibility to utilize multiple CPUs is added to DAMMIF. The application is available in binary form for major platforms.
Small-angle x-ray scattering (SAXS) of biological macromolecules in solutions is a widely employed method in structural biology. SAXS patterns include information about the overall shape and ...low-resolution structure of dissolved particles. Here, we describe how to transform experimental SAXS patterns to feature vectors and how a simple k-nearest neighbor approach is able to retrieve information on overall particle shape and maximal diameter (Dmax) as well as molecular mass directly from experimental scattering data. Based on this transformation, we develop a rapid multiclass shape-classification ranging from compact, extended, and flat categories to hollow and random-chain-like objects. This classification may be employed, e.g., as a decision block in automated data analysis pipelines. Further, we map protein structures from the Protein Data Bank into the classification space and, in a second step, use this mapping as a data source to obtain accurate estimates for the structural parameters (Dmax, molecular mass) of the macromolecule under study based on the experimental scattering pattern alone, without inverse Fourier transform for Dmax. All methods presented are implemented in a Fortran binary DATCLASS, part of the ATSAS data analysis suite, available on Linux, Mac, and Windows and free for academic use.
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.
Assessing similarity between data sets with the reduced χ(2) test requires the estimation of experimental errors, which, if incorrect, may render statistical comparisons invalid. We report a ...goodness-of-fit test, Correlation Map (CorMap), for assessing differences between one-dimensional spectra independently of explicit error estimates, using only data point correlations. Using small-angle X-ray scattering data, we demonstrate that CorMap maintains the power of the reduced χ(2) test; moreover, CorMap is also applicable to other physical experiments.
•Popularity of SAXS/WAXS for structural analysis has gained substantially.•Remarkable developments in hardware and software allow for high-throughput studies.•SAXS became an integral part of hybrid ...approaches to analyze complexes.•Flexible systems are quantitatively characterized by ensemble analysis approach.•WAXS permits one to monitor subtle structural changes.
The advances made in small and wide angle X-ray scattering over the past decades have had a large impact on structural biology. Many new insights into challenging biological probes including large and transient complexes, flexible macromolecules as well as other exciting objects of various sizes were gained with this low resolution technique. Here, we review the recent developments in the experimental setups and in software for data collection and analysis, specifically for hybrid approaches. These progresses have allowed scientists to address a number of intriguing questions which could not be answered with other structural methods alone.
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