Hello and welcome to the Membrane Structural and Functional Biology Group. My name is Martin Caffery. This is another in a series of JO of articles on crystallizing membrane proteins for structured determination using the in meso or lipic cubic phase method.
In the first article, JoVE 1 7 1 2, we demonstrated how the protein laden cubic phase is prepared and how the viscous and sticky misa phase is dispensed manually into glass sandwich plates for crystallization trials. This video article will describe methods for using an in meso crystallization robot. A robot offers the advantages of speed, accuracy, precision miniaturization, and being able to work for extended periods under uncomfortable conditions such as in the dark or at low temperatures.
Robots when used properly can greatly improve the productivity of membrane protein structure and function research. In this video article, we demonstrate the use of three commercially available robots. The first robot was developed in the membrane structural and functional biology group, and is described in detail in reference one.
The other two have just recently become available and are included here for completeness. Preparing the crystallization plate setting up to do a crystallization trial robotically begins by preparing the base plate of the glass sandwich crystallization plate described in detail in reference to the base plate must first be siloized and the perforated double stick spacer that creates the wells must be applied to the plate. The materials needed for this are shown here.
They include a glass crystallization base plate, a perforated double stick spacer izing solution, a roller or brayer paper tissues, and a beaker of UE water. The plate is placed on a paper towel on a bench top. A few drops of silent solution are applied and then a distributed evenly over the plate surface with a paper tissue.
The plate is washed of excess izing solution by immersing it fully in a beaker of water. The plate is removed from the beaker placed on a dry paper towel. Siloized side up and its exposed surface is dried by wiping lightly with paper towels.
Next, the protective cover on the perforated double stick spacer is removed and the spacer is applied to the siloized surface of the glass plate. It is important that the spacer is positioned properly on the base plate with the upper left hand corner and the two adjacent sides of the spacer and the plate coincident. A roller or brayer is used to flatten out the spacer on the base plate and to create a tight seal between them.
Parenthetically, we note that siloized plates complete with perforated spacers are available commercially as described in the reference section. Having prepared the crystallization base plate, we next move on to setting up and to using the in meso robot. As noted in what follows, we demonstrate the use of three in meso robots.
In all cases shown the robots are set up and used in a temperature controlled room set at 20 degrees Celsius in meso robot one, the first in meso robot to be demonstrated in this video. Article was developed in the PI's laboratory and is now available commercially. Full details can be found in the reference section.
The materials and the equipment required to set up a crystallization trial robotically are shown here. They include a laboratory notebook, protein laden mease, freshly prepared as described in Jo of Article 1 7 1 2 and housed in a Hamilton syringe. Siloized base plates with spacers and matching cover glass, water bottle and tissues, and the in miso robot.
The first view is of the robot with its deck clear. The next view is of the robot complete with humidifier park station, crystallization base plate and deep well block containing precipitant solutions. We begin the setup by placing the base plate on the deck of the robot.
It has its own platform, clearly marked with well positions for purposes of base plate alignment. The plate should be clearly labeled for identification purposes. The 96 well block containing precipitant solutions is secured in its own platform on the deck of the robot next to the crystallization plate.
The block is opened by carefully peeling off its plastic ceiling cap. The humidity stream from the humidifier is directed to flow over and across the base plate on the deck of the robot Setting up the robot. The robot is initialized by following the manufacturer's instructions.
We will not demonstrate this in any detail here because the exact protocols will change over time. Essentially what happens during initialization is that the mease dispensing arm on the robot is provided with reference positions in three orthogonal directions, x, Y, and Z.Next, the protein laden meza phase in a Hamilton syringe complete with dispensing needle is attached to arm one of the robot full details of how to prepare the meza phase and the syringe are described in Jo of Article 1 7 1 2 and in reference one, the needle tip of the dispensing syringe must now be aligned with the bottom and the center of the uppermost well in the left hand corner of the crystallization plate. This corresponds to well identifier A one aligning with the bottom of well A one as the first order of business.
This is done by I adjusting the height or Z coordinate after dispensing arm while watching how close the needle tip comes to the bottom of the well. That the tip is just touching the bottom of the well can be judged by moving the plate by hand from side to side on the platform while ever so slightly adjusting the height of the dispensing arm, the Z coordinate of the dispensing arm where the tip contacts the plate is entered into the computer dispensing is programmed to take place with the needle tip several micrometers from this reference Z position, which corresponds to the surface of the base plate. The robot is next activated to go through a series of steps prompted by the user to position the tip of the needle at the center of well A one.
The reference XY coordinate of the dispensing arm where the tip is centered is stored in the computer controlling the robot. It is used to define the center of the remaining wells on the plate. Having set these reference coordinates in the computer, the mease dispensing arm is instructed to return to its starting position, priming the syringe.
The dispensing syringe must now be primed. This is done to ensure that fresh mease fills the dispensing needle and that the first well on the plate receives its full complement of mease. There is a priming command in the program that runs the robot activating.
It causes the plunger in the syringe to advance stepwise under user control. Whilst this is happening, the tip of the needle is examined for mease to appear extruded meza phase is removed with a tissue. This completes the priming process running the robot.
The robot is now ready to run in automatic mode starting the run. Robot XAP program initiates a round of flushing steps where the precipitant dispensing tips on arm two of the robot are washed and are prepared to aspirate precipitant solutions from the deep well block activating the dispense function on the robot sets in motion, the sequential loading of wells with protein laden mease first eight wells at a time, followed by precipitant solutions Between deliveries. The mease dispensing arm returns to a parking position where the end of the needle is placed in a moistened sponge to prevent the mease at the tip from drying out.
Most commonly, the values used in crystallization trials carried out in the membrane. Structural and functional biology group are 50 nanoliters of mease and 800 nanoliters of precipitant solution. The program running the robot in this demonstration has been set up so that the two arms move simultaneously.
In this way, it is possible to complete the loading of a 96 well plate with mease and precipitant in five minutes. Immediately the plate is filled and the robot arms have returned to their park positions. The plate is removed from the deck of the robot and placed on a flat solid surface.
A cover glass is laid down gently on top of the base plate in contact with the upper sticky surface of the spacer. It is important to ensure that all 96 wells are fully covered, that the cover glass is align square with the base plate and that none of the cover glass extends beyond the footprint of the base plate. A roller or brayer is used to create a tight and a uniform seal between the cover glass and the spacer.
The plate should be inspected at this stage to be sure the setup went as it should. What one is looking for is a fried egg on a pan appearance. In each well, the yoke of the egg corresponds to the meza phase.
The white of the egg is the precipitant solution, and the edge of the pan is the perimeter of the crystallization. Well, ideally, the three are concentric. The plate is now ready to be placed in a temperature controlled storage area for crystal growth.
If no more plates are to be set up, the precipitant block should be removed from the deck of the robot, properly sealed and put back into storage. The syringe containing the mease is removed from the dispensing arm of the robot, dismantled its various parts, including dispensing needle and ferial. Washed carefully with methanol and dried in preparation for next Use In Meso robot two mosquito LCP.
This next video clip shows the mosquito LCP robot that can be used in the setting up of in meso crystallization trials. It incorporates a similar meph phase dispensing system to that described previously in this article. For robot one, however, the precipitant is dispensed using disposable tips.
The instrument itself is shown here. It includes a deck upon which the crystallization plate and precipitant block set a windowed chamber for control of humidity. In this view, the windows on the humidity chamber have been opened a spool with dispensing tips, a mease dispensing arm, a laser-based sensor for establishing the location of the meph phase dispensing needle.
A raised platform upon which the crystallization plate sits a sunken platform for holding the precipitant screen block. In this sequence, the robot has seen going through an initializing sequence of actions when the instrument is turned on. Programming software is used to set up the instrument for working with particular crystallization plates and precipitant blocks, and to dispense set volumes of mease and precipitant in a specified sequence.
Such details are provided by the manufacturer and will not be shown here. Using the robot to set up a crystallization plate begins as follows, A Hamilton syringe complete with needle and filled with protein laden mease is prepared as described above under in meso robot one and in Jo of 1 7 1 2. The syringe is clamped in position under dispensing arm of the robot.
The X and y coordinates of the needle on the dispensing syringe are established using the deck mounted laser sensor. This is an important step that is required to ensure that the mease is dispensed accurately at the center of each well on the crystallization plate. With the touch of a button on the control panel, the syringe is primed to expel mease from the tip of the needle.
Extruded mease is then removed with a tissue in preparation for using the robot to load wells before mounting the crystallization plate on the deck of the robot. The surface of the platform is moistened with a few drops of water to hold the plate in place. By capillary, the protective cover is removed from the plate and the plate is placed on its platform firmly to ensure good adherence.
There are three fiducials on the platform over which the three corner wells are centered soon to ensure proper positioning of the plate with respect to the dispensing needle tip, the instrument is now ready to enter a setup cycle. This begins with a second priming step where Mease is dispensed onto a small glass plate immediately before moving on to load the plate with fresh mease. In this sequence, we see the robot first loading with mease, a vertical column of eight wells on the plate, And then using the disposable tips to dispense precipitant solution on top of the mease bolus.
This process is repeated 11 times along the length of the plate until all wells are filled. The entire process takes about five minutes to complete. In this particular video, clip 50 nanoliters of Mease and 800 nanoliters of precipitant solution were dispensed into each well.
The filled plate is removed from the platform of the robot placed on a bench. The cover glass is applied and the plate is sealed with a bra as described above under in miso robot one. The plate properly labeled can now be placed in a temperature controlled environment for crystal growth in Meso robot three, the Griffin LCP.
This final video clip shows the Griffin LCP robot in use setting up in meso crystallization trials. The Griffin incorporates a similar meph phase dispensing system to that described above for robot one. In this case, however, all 96 precipitant solutions are dispensed simultaneously.
The instrument itself is shown here. It includes a deck on which the crystallization plate and precipitant block sit, a mease dispensing arm and a Phoenix 96 tip head. For dispensing precipitant solutions, the robot in action is shown in this final video sequence.
The 96 tip head is first loaded with precipitant solution. A user defined delay is provided to facilitate aspirating viscous precipitants. The MEA phase is dispensed sequentially into the 96 wells in a process that takes about one minute to complete.
Lastly, the 96 tip head dispenses all 96 precipitant solutions simultaneously on top of the MEA phase boluses in the crystallization wells. The entire plate loading process is completed in two minutes. The filled plate is removed from the deck of the robot and the plate is sealed with a cover glass and placed in storage under controlled temperature conditions for crystal growth.
The next steps in the overall process of structured determination by macromolecular crystallography are to harvest and cryo cool the crystals, and to record and process x-ray diffraction from them. These topics are covered in separate job articles in this series.I.
