Towards a better understanding of macromolecular X-ray structures

• Applicant: Dr. Andrea Thorn
• Subject Area: Structural Biology
• Term: from 2019 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 420163647

For over 50 years, X-ray crystallography has been the primary method to determine the structures of biological macromolecules. However, the atomic structure is not the direct outcome of the experiment: prior knowledge of macromolecular structures and experimental methods is used in most steps between measurement and the final model. Consequently, our structures are only as good as our fundamental understanding of their nature - and our ability to express this in structural models and the methods employed.The discrepancy between X-ray data and structural models is usually given as a percentage called the R value. While small molecule structures routinely reach R-values of 5%, macromolecular structures typically are at 20%-25%. These relatively high discrepancies between processed data and structural model are the main reason why some relevant biological questions - for exmple whether a ligand is bound - cannot be answered and why some structures cannot be solved at all, for example membrane proteins and large complexes, where often only low-resolution data are available.In macromolecular crystals - as opposed to small molecule crystals - typically half of the volume is occupied by disordered solvent, which affects the ordered macromolecules so that they adopt slightly different conformations dependent on the local environment. Current models do not describe this disorder very well. Further improvement is hindered by the fact that we use model phases to generate maps. Since this introduces a strong model bias, we cannot observe what we do not anticipate. In this project, model-bias free maps, calculated from purely experimental phases, will be used to overcome this challenge and to provide an improved model of macromolecular crystal structures. Parallel to this we will improve the quality of X-ray data. recently developed program, AUSPEX, which indicates pathologies in crystallographic data. Currently, it is mainly used to detect ice rings, but in this project it will be expanded to show users further problems during data collection and processing and improve our understanding of the actual experiment. In addition, we have initiated an AUSPEX web server for fast and easy access for crystallographers online.With these two objectives - improved structural models for macromolecular crystals and better X-ray data - we will be able to lower R-values and consequently, too see more detail in new as well as existing structures. These improvements will enable structural biologists worldwide to adress more challenging biological problems.

• DFG Programme: Research Grants