Small-angle X-ray scattering from macromolecular solutions
Visualising a decoy protein of a herpes virus at work
The Epstein Barr Virus (EBV), a member of the herpes virus family, is a global human pathogen causing a diverse range of diseases. A multidisciplinary study using synchrotron radiation, electron microscopy, biophysical and cellular methods (Elegheert et al., Nat Struct Mol Biol. 2012 19:938-47) revealed at the molecular level how the virus can deactivate the alert system of the body’s immune defence. The EBV secretes a protein BARF1 that neutralises a human colony-stimulating factor 1 (hCSF-1). It is shown that the flexible BARF1 protein locks the dimeric hCSF-1 into an inactive conformation, rendering it unable to perform its signalling function. The novel mechanisms have implications for the development of therapies and drug compounds. In the figure, the crystal structure of the BARF1-CSF1 complex (top left) is validated by SAXS data (bottom left), whereas the crystal structure of the free BARF1 (blue cartoon, top right) does not agree with SAXS (bottom right) and was refined using normal mode analysis (red cartoon, top right).
The Svergun group places special emphasis on hybrid methods combining SAXS with X-ray crystallography, NMR spectroscopy, and electron microscopy to improve resolution and cross-validate structural models.
Previous and current research
Small-angle X-ray scattering (SAXS) reveals low resolution (1-2 nm) structures of biological macromolecules in close-to-native solutions for an extremely broad range of sizes – from small peptides to huge macromolecular machines – and in variable conditions. For many complicated biological systems which may be flexible or have a dynamic nature, SAXS is the only method capable of providing structural information.
Recent experimental and methodical developments have significantly enhanced the resolution and reliability of the SAXS-based structural models. This versatility and universality – and the fact that it does not need crystals to characterise the structure – makes SAXS an ideal tool for systems structural biology, and the last decade saw a renaissance of biological SAXS worldwide.
Our group leads the development of novel computational methods for constructing 3D structural models from the scattering data. Special attention is given to the joint use of SAXS with other structural, biophysical and biochemical techniques including: crystallography, NMR spectroscopy, electron microscopy, neutron scattering, and bioinformatics. We developed ATSAS, the world’s most popular biological SAXS analysis program suite, which has been downloaded by over 7000 users, and we continue to provide the scientific community with novel approaches.
We run a brand new high-brilliance synchrotron beamline P12 at DESY’s third-generation storage ring, PETRA III. The beamline, commissioned in 2012, is optimised and dedicated to biological solution SAXS. P12 is equipped with a robotic sample changer, utilising 96 well plates for rapid automated experiments. It possesses a data analysis pipeline for building structural models online, with FedEx-style and remote data access options. As of 2012 we also offer an in-line size exclusion chromatography setup, with biophysical sample characterisation using a triple detector Malvern box.
Most of the external users of P12 are seeking collaborative projects where the SAXS group members help not only with data collection but also with modelling. In numerous exciting applications, SAXS is employed to study overall structural organisation and flexibility of individual macromolecules and complexes (see figure) and conformational transitions (for instance upon ligand binding). It is also used to characterise oligomeric mixtures, flexible systems and intrinsically unfolded proteins, hierarchical systems, and many other objects of high biological and medical importance.
Future projects and goals
- Further development of novel methods and approaches for the reconstruction of tertiary and quaternary structure of macromolecules and complexes from X-ray and neutron scattering data.
- Hybrid applications of SAXS with crystallography, NMR, electron microscopy and other methods and the use of bioinformatics to construct and validate SAXS-based models.
- Participation in collaborative projects at the P12 beamline employing SAXS to study the structure of a wide range of biological systems in solution.
- Further improvements of the capabilities of P12, including complete automation of biological SAXS experiments and data analysis, on-line FPLC, and time-resolved scattering setups.