New crystallization strategies to fast-feed structure-guided pharmacological development – Applications to large biological assemblies involved in RNA metabolism

The elucidation of three-dimensional structures of molecular machines that control cellular physiology is necessary for the understanding of the mechanisms of life and for the development of rational screening tests for pharmaceutical applications. Due to the large size of these biological entities and the high resolution which is sought, X-ray crystallographic structure determination is the method of choice. Obtaining crystals of biological complexes however remains difficult and is the bottleneck to this method. In this project, we have applied sophisticated crystallization strategies to a hitherto intractable problem: crystallising a molecular motor, namely the bacterial transcription termination factor Rho from Mycobacterium tuberculosis. Rho is a ring-shaped hexameric helicase targeting transcriptional complexes and R-loops, and regulating RNA metabolism in a variety of ways. The first crystals of M.tub. Rho have been obtained, which however should now be optimised to reach an X-ray diffraction resolution sufficient for full three-dimensional structure determination. In addition, we have developed a theoretical model describing the varying usefulness of ions at different positions in the Hofmeister series, according to thermodynamic properties of the crystallizing protein.