In this video article we have demonstrated how to use a robot to automatically set up in meso crystallization trials in 96-well glass sandwich plates using a protein-laden lipidic mesophase. The robots used in this work were specially designed to include a positive displacement glass syringe for accurate and reproducible delivery of nanoliter volumes of the viscous and sticky mesophase, as originally described7.
Accuracy and precision are important features of a robot. However, these characteristics are only as good as the degree to and regularity which robot performance is evaluated and calibration is performed. It goes without saying that performance of the robot should be evaluated while individual plates are being set up. It is not appropriate to assume that the robot will function flawlessly and to leave the robot to run unattended. The attentive and observant operator should be in a position to note by a sound or an appearance when something is not working properly and to correct it immediately. Additionally, each plate should be carefully inspected by eye for uniformity of well contents as soon as the plate is sealed and before it is put away for crystallization trials. This should only require a few seconds to perform and can be done while the next plate is being loaded. Noticing, for example, that particular wells are not properly filled may highlight the fact that a certain precipitant dispensing tip is malfunctioning. Should delivery of mesophase be seen to be irregular, the relevant faulty item would need to be corrected immediately. By noting these issues and making the relevant adjustments during set up will save time and materials, including valuable lipid and membrane protein. If an imager is used to track crystal growth, accuracy and precision can also be monitored during imaging. For example, systematic problems with drop or bolus location by the imager indicate that something is not right and that corrective action somewhere upstream in the protocol is needed.
In the interests of reliable performance therefore the robot must be calibrated on a regular schedule and as needed. Calibrations should include volume of precipitant and mesophase delivered as well as bolus and precipitant placement in the well. Obviously, wherever possible calibration should be done using volumes and materials similar to those that will be used in trials conducted in the period covered by the quality control exercise.
As important as calibration is so too is having in stock an adequate supply of robot parts and supplies. Catastrophic and unexpected failures, blockages and crashes can and do occur. In such an event, having available a replacement precipitant delivery tip, for example, might mean that an extremely valuable membrane protein preparation is used as it should and is not wasted.
Some of the advantages of a robot include the fact that it will work essentially continuously and that it does not suffer from or 'complain of' fatigue. A robot can also be used under conditions that are not considered human-friendly, such as in the dark, under controlled lighting and environmental conditions, and at extremes of temperature. The robots demonstrated in this article were all used under ambient conditions at ~20 °C. However, there are proteins and projects that require non-ambient temperatures, controlled light11,12 and an oxidizing or a reducing environment13. All of these can be catered for, with relative ease, when a crystallization robot is used.
In an earlier JoVE video article we demonstrated how crystallization trials by the in meso method are set up manually 1. The minimum volume of mesophase that can be dispensed reliably by hand is limited by the visual acuity and steady handedness of the person setting up the trials. In our experience, volumes of mesophase as low as 100 nl are readily handled. We know of one lab where the default volume dispensed manually is about 40 nl. However, considerably smaller volumes are possible using a robot. Separately, we have shown that mesophase volumes as low as 550 picoliters can be dispensed robotically14. From that work it was clear that further miniaturization by robot is possible which, if implemented, would lead to a large reduction in the amount of valuable membrane protein needed to carry out a crystallization trial.
In this video article, three commercially available robots were used to demonstrate high-throughput membrane protein crystallization using lipidic mesophases. The first of these was developed in the MS&FB Group based on our experience setting up trials manually, as described in JoVE 17121. This is the instrument we have most familiarity with and the bulk of the current article is devoted to its use. The other two robots were on demonstration in the MS&FB Group at the time the article was written and the relevant footage is included here in the interests of completeness. All three robots use very much the same mesophase dispensing system, the essential feature of which is a positive displacement glass syringe5,7. They differ most significantly with regard to precipitant delivery. Robots 1 and 2 can dispense precipitant simultaneously into 8 wells, a single column on a crystallization plate at a time. In contrast, Robot 3 dispenses all 96 precipitant solutions in a single action. Robot 2 is the only instrument with disposable precipitant dispensing tips. There are pros and cons associated with the different instruments that depend on the particular application; these will not be elaborated on here. Suffice it to say that all three work and have produced crystals of membrane proteins by the in meso method.
The next steps in the overall process of structure determination by macromolecular crystallography are to harvest and to cryo-cool crystals from plates set up as described in this video article and to record and process X-ray diffraction from them. These topics are covered in separate JoVE articles in this series1,15.