Despite having an optimal sample for crystallization, obtaining crystals can be an arduous and unpredictable goal. The most important approaches to overcome this bottleneck were the discovery of appropriate precipitants, new methods of preparation of macromolecule samples, new methods of carrying out crystallization experiments but above all the increase of crystallization efficiency through automation.
The latter approach is based on the awareness that the intrinsic impossibility to predict the conditions that favor crystal formation is overcome by screening a variety (tens) of constructs of a target of interest against a large number (hundreds) of possible precipitating agents. Crystallographers are therefore forced to design and conduct a large number of experiments (about 500-1000 single sample tests) before identifying favorable conditions for crystal formation. Setting up crystallization experiments by hand is not only tedious, but also involves inefficient use of qualified personnel and/or students and, beyond a certain number of conditions, it is only approachable by means of robotic equipment.
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.
DESCRIPTION OF THE EQUIPMENT
The crystallization robot is an instrument that allows the automated production of crystallization plates. In a crystallization plate, 24, 48 or 96 different crystallization conditions are tested simultaneously. For each condition a micro / nano volume of a solution containing the target protein is mixed with a suitable amount of crystallization solution present in the single well of the plate. Subsequently each condition is sealed and, by vapor diffusion, the concentration of the individual components of the drops thus obtained varies very slowly over time (from a few days to several weeks) until in one or more conditions the formation of protein crystals is observed. Normally, to obtain initial crystallization conditions it is necessary to test from 200 to 1000 different conditions for protein sample (about 2-10 plates of 96).
The use of a crystallization robot reduces the time needed to setup the crystallization trial of 100 times and the protein amount of 10 times (manual setup: 12conditions/h ; automatic setup: up to 1000 condition/h). In other words, using a crystallization robot allows to sample the crystallization space of a protein (960 tests) in about an hour, whereas it would require 2 weeks of work to do it by hand. For this reason, to remain competitive with international laboratories, the use a crystallization robot is mandatory.
The robot use multi-hole dispensing tips, which have several independent channels to deliver small volumes. Each channel provides a different solution. The solutions do not mix in the tip: they mix in the drop after they have been dispensed; this means that no dead volume remains.
The robot is equipped with the LCP module has the following features:
Soluble Protein Dispensing:
• can accurately and reproducibly set up sitting drop crystallization experiments. The same system can set up nano-drops for screening (eg 100 + 100 nl) or micro-drops for optimization and data collection (e.g. 2 + 2 μl). For crystal harvesting and soaking experiments a higher volume (up to 8+8μl) is possible.
• can set up hanging drop experiments with 24-well Linbro or SBS plates and glass (or plastic) cover slides. Up to 5 drops can be dispensed per slide, and the volume of protein, reservoir solution and additive (such as seed stock) in each drop can be varied at will.
• wastes virtually no protein sample or seed-stock - only 0.5 μl are lost for a 96-well plate. Contact dispensing means that almost no protein remains in the tip at the end of the experiment. Also, since only one (multi-channel) tip is used, all of the protein for an experiment can be placed in a single PCR tube, which also reduces waste.
• can perform microseed matrix screening (MMS) experiments using a simple one-pass routine. Contact dispensing enables a suspension of crystal seeds to be dispensed, without risk of blockages.
• possesses powerful but user-friendly software for designing screening, MMS microseeding, additive, 2-d grids (protein is varied against precipitant or additive), Combinatorial (see below), Additive Scatter (see below), microbatch-under-oil, hanging drop and sitting drop experiments. No programming or scripting by the user is required.
• The software allows individual wells to be selected for dispensing. For example, wells that gave heavy precipitate can be ‘cherry-picked’ with a mouse and repeated with a lower protein concentration.
• can use virtually all crystallization plates, including non-SBS compatible plates. The system is extremely flexible with regard to plates. New plates can be added as required by the user.
• is not dependent on consumables (other than crystallization plates).
• can perform Combinatorial experiments in which ingredients can be reshuffled and/or appropriate dilutions of seed stocks can be found. Additive Scatter experiments are similar except that the seeds/additives are scattered evenly around the plate.
• can dispense a vapor diffusion experiment and a microbatch-under-oil experiment in parallel.
• can fill reservoirs for simple optimization experiments.
Membrane protein dispensing using Lipidic Cubic Phase (LCP):
• LCP functionality sets up crystallization experiments with LCP, dispensing to sandwich plates, sitting drop plates or Linbro plates etc. with cover slides. The volume of the LCP bolus can vary from around 5 nl to 8 μl.
• Two dispensing methods are available – flattened boluses are generally used for volumes below 200 nl, with conical boluses for larger volumes.
• LCP screening, additive screening and combinatorial optimization experiments are performed.
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.