Introduction
Determining X-ray structures has become more automated, faster, and more accessible since the commencement of protein crystallography. The initial step for crystallization is still an erratic and time-consuming process. The protein structures can be solved in a couple of hours, and crystallization has grown into a significant bottleneck of X-ray crystallography. (Stock et al. #)
-
Preparation Of Crystallization Plates
- Design And Operation
A robot with a set of eight tips for simultaneous transfer of solutions is required for re-formatting the commercially obtained crystallization reagents into the 96-well crystallization plates. The spacing between5 ml test tubes, which contains reagents, is much wider as compared to the spacing between wells in the plate. The Tecan tips with adjustable distances are needed. The cost and plastic consumption are reduced by using fixed tips that should be washed between the steps as prevention for cross-contamination. The setup requires a device for intermediate plate storage to achieve minimal manual handling. An automated plate sealer is used, and a robotic arm is used to transfer plates. Some reagents are heat-sensitive, so the choice of plate sealers is restricted to only those that use adhesives in place of heat. The system meeting the requirements is the Tecan Genesis 150 liquid handler. The system is supplemented with an adhesive plate sealer. It does not require any changes from factory design or any custom-made parts.
- Optimization Of Handling The Liquid
The aspiration steps and dispensing steps have to be optimized along with the physical movement of tips to ensure accurate handling of viscous solutions. The robot provides the definition of different classes of liquid, such as very viscous to volatile. This requires the optimization of various parameters like the aspiration speed, size of the air gap, volume delay that occurs after aspiration, and the same parameters for dispensing. The pipetting is optimized for the most viscous solutions and used this class for all reagents since the crystallization reagents from commercially acquired kits are not ordered on the basis of their liquid properties, and ordering again can create logistic issues. The most severe problem for the dispensing is to ensure that mixing of system liquid with crystallization reagent does not occur while dispensing as this can cause an irregular dilution of crystallization reagents. The only means of avoiding this is to aspirate a large excess volume, around 40 percent more than the amount required for dispensing. With this aspirated volume, four plates can be dispensed in a multi-dispensing mode, and each of eight tips dispenses 4×85μl. Four plates are the highest number that is possible to be handled at one time as the main priority is to keep the opening times for each plate under 15 minutes. Once the four plates are filled, the robotic arm is used to carry each plate to the plate sealer and then bring it back to the carousel. The initial idea for a long-term means of plate storage is to store them at -20°C. This will prevent the deterioration and microbial growth of less stabilized compounds.
-
Setup Of Nanolitre Crystallization Experiments
A robot is required in this step to transfer nanolitre amount of reservoir solution into small crystallization wells that are attached to the reservoirs, which could add the same amount of protein to each well. The robot needs to be very precise and fast for even a very small amount of volume. The Cartesian Technologies PixSys 4200 SynQuad liquid handler can fulfill these requirements. This technology makes use of a combination of a syringe and solenoid value that is able to dispense small quantities of liquid very quickly without even touching the surface of the plate.
-
Optimization Of Crystalline Conditions
Optimization of initial crystallization conditions became increasingly time-consuming after robots were in operation for preliminary screens. Initiating a manual optimization process using a robot is rather tricky because of the wide range of concentrated stock solutions, which need to be pipette in the crystalline plates in a broad range of volume.
-
Conclusion
The automated crystallization setup has entirely replaced the manual handling of the liquids. Even though the crystallization of biological macromolecules is still unpredictable, the automation process has significantly decreased the amount of effort and time required in the process.
Bibliography
Stock, Daniela, et al. “Robotic nanolitre protein crystallisation at the MRC Laboratory of Molecular Biology.” MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK, vol. 88, no. 3, 2005, pp. 311-327. https://doi.org/10.1016/j.pbiomolbio.2004.07.009.
