The structure of macromolecules is a crucial information to precision medicine, biotechnology and drug design. The structure of molecular targets allows to understand the fine mechanisms of cell physiology and pathology. Academic and for-profit organization are racing to determine the structure of all Covid-19 proteins and of their complexes with host interactor to design new drugs and therapeutic strategies.
Sapienza is at the forefront of structural biology and it has been active, with its Structural Biology group, giving an internationally recognized contribution to the understanding of pathological phenomena at the molecular level, of protein structural dynamics and of protein usage for biomedicine and biotechnology.
Since its inception, the Dept. of Biochemistry of the Sapienza University, in collaboration with the CNR Institute of Molecular Biology and Pathology, has deposited over 200 protein structures in the Protein Data Bank and published over 300 papers.
The main methodology in the field is X-ray crystallography, based on diffraction by protein crystals, it is of vital importance to utilize high-throughput equipment to maximize crystal yield. The acquisition of a latest generation crystallization robot to replace the present obsolete and unrepairable equipment will allow the continuation of a high-profile research activity and expand its potential. It will allow the Structural Biology group to provide a service to other Depts of Sapienza and/or external parties, including Companies. This equipment will be unique at Sapienza, in Rome and in the region Lazio, serving all central Italy.
Furthermore, it will create a synergy with other equipment for the biophysical characterization of proteins (eg SAXS, electron microscope) and it will continue to support grant application in Sapienza for Departments involved in molecular biology, drug design, biomedicine and physiology, strengthening the competitiveness of Sapienza at the European and national level.
The crystallization of biological macromolecules is based on an extended set of principles, experiences and ideas and there is no complete theory, or a fundamental database to follow. As a result, crystal growth is largely empirical in nature and it requires patience, perseverance and intuition. Complicating the entire process, in addition to limited understanding of the phenomena involved, is the complexity of the macromolecules being studied: in the case of viruses or enzymatic complexes the possibilities of conformations, interaction and mobility are almost incalculable.
To date, macromolecules crystallization is based on a "trial and error" approach in which we first try to find the conditions in which the initial crystals grow and then optimize the single variables to obtain high quality crystals, useful for structural determination. This is achieved by carrying out numerous tests, evaluating the results and using this information to improve conditions in subsequent cycles of crystallization trials. Since the number of variables is very high, experience, intuition and, above all, automation are fundamental aspects.
Moreover, robotics is able to manage liquids efficiently, to combine them accurately in new formulations and to generate a large number of experiments, using small volumes (
For about ten years, a Phoenix robotic station (Art Robbins) has been available for collaborations and for users of Sapienza at the Department of Biochemical Sciences. The crystallization robot is positioned in a room with controlled temperature and humidity dedicated exclusively to crystallization. This crystallization robot is now experiencing malfunctions that cannot be eliminated, such as the lack of calibration of some of the dispensing channels that do not allow to set the mixing volumes, leading to a scarce experimental reproducibility. These faults cannot be repaired since the equipment is no longer in production and all interventions have to be custom made, at a prohibitive price and are not guaranteed by the manufacturer.
Moreover, its operating system is now obsolete and it does not provide the possibility of performing crystallization experiments with particular and innovative techniques such as seeding (i.e .the technique that involves inserting crystallization micronuclei from the outside to favor the process of nucleation) and crystallization in oil. The seeding procedure is now considered a winning strategy both to increase the probability of obtaining crystals in the initial screening phase and to improve its quality in the optimization phase.
Moreover, the possibility of using this technique is particularly advantageous if one wishes to characterize protein-ligand complexes or in projects aimed at the structural characterization of protein-inhibitor complexes particularly useful in projects concerning the rational design of drugs. On the other hand, the possibility of performing crystallization in oil in a robotic manner would allow the use of an additional technique for the crystallization of proteins, significantly increasing the probability of success.
While the number of structures resolved thanks to biocrystallography continues to grow exponentially reaching almost the number of 140000 in the protein structure database (Protein Data Bank) the structural and functional understanding of membrane proteins is strongly lagging behind, mainly due to the difficulties related to solubilization and to the production of crystals useful for diffractometric analysis.
Indeed, membrane proteins are generally crystallized not in aqueous solutions such as soluble proteins but in lipid-containing solutions through a technique known as Lipidic Cubic Phase (LCP).
This technique exploits the advantage of crystals being grown in an environment similar to that of biological membranes. The crystals obtained with this technique contain a lower percentage of solvent and are more ordered and of better diffractometric quality than those traditionally obtained from detergent solutions. This crystallization process requires an instrumentation not currently available in the Department of Biochemical Sciences.
The equipment we plan to acquire in the context of the present call would allow us to carry out lipidic cubic phase crystallization experiments, thus opening new perspectives and projects to the scientific community of the Sapienza University.
Therefore, the last generation crystallization robot proposed is essential to keep Sapienza competitive in the field of structural biology both in terms of scientific productivity and the ability to attract national and international funding and it will considerably expand its scientific goals and perspectives.