Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

1. Preparing the Crystallization Plate

Setting up to do a crystallization trial robotically begins with the preparation of the base-plate of the glass sandwich crystallization plate (Figure 2), described in detail in Reference 2. The base-plate must first be silanized and the perforated double-stick spacer that creates the wells, must be applied to the plate. The materials and supplies needed for this are itemized under Materials.

1. Place the plate on a paper towel, apply a few drops of silanizing solution and distribute it evenly over the plate surface with a paper tissue.
2. Remove excess silanizing solution by immersing the plate in a beaker of water.
3. Take the plate out of the beaker, place it on a dry paper towel, silanized side up, and dry its exposed surface by wiping lightly with a paper towel.
4. Peel off the protective cover on the perforated, double-stick spacer and apply the spacer, sticky side down, to the dry, silanized surface of the glass plate. Be sure 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.
5. Use a roller or brayer to flatten out the spacer on the base-plate and to create a tight seal between them.

Pre-assembled plates are commercially available (HR3-151, Hampton Research; MD11-55-100, Molecular Dimensions; and LCP96T-UVP70U, Swissci). For cost reasons, we prefer to assemble our own plates following the protocol above.

2. In Meso Robot 1

The first in meso robot to be demonstrated in this video article is the one currently in use in the MS&FB Group (Figure 3). Full details of the robot can be found under Materials and in the Reference section⁵. The materials and equipment required to set up a crystallization trial using the robot are itemized under Materials.

1. Place the base-plate with spacer prepared in Section 1 on the deck of the robot. It has its own platform marked with well positions for purposes of base-plate alignment. The plate should be labeled for identification purposes.
2. Secure the 96-well precipitant block in its own platform on the deck of the robot next to the crystallization plate.
3. Open the block carefully by peeling off the plastic sealing cap.
4. Turn on the humidifier and direct the stream of humid air over and across the base-plate on the deck of the robot. The humidifier has been shown to reduce evaporation of the precipitant solution and to enhance the reproducibility across the crystallization plate⁵. It can be turned on prior to set-up to pre-humidify the area when ambient relative humidity is low, or otherwise, just before the dispensing of mesophase commences.
5. Initialize the robot following the manufacturer’s instructions. Instrument specific instructions will not be described here in any detail because exact procedures will change over time. Essentially, what happens during initializing is that the mesophase dispensing arm on the robot is provided with reference positions in three orthogonal directions X, Y and Z.
6. Attach the protein-laden mesophase in a Hamilton syringe, complete with dispensing needle, to Arm 1 of the robot (Figure 3). Full details of how to prepare the mesophase and how to load it into the syringe have been described in References 3,7-10.
7. Remove the upper protective cover from the spacer on the crystallization base-plate and align the wells on the plate with fiducials on the base-plate platform. Note, for the purpose of this video, the protective cover was left in place to aid the Viewer during the alignment step.
8. Align the needle tip of the dispensing syringe with the bottom of the uppermost well in the left hand corner of the crystallization plate (Well A1). This is done by eye adjusting the height or Z-coordinate of the 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 from side to side on the platform whilst ever so slightly adjusting the height of the dispensing arm.
9. Enter into the computer the Z-coordinate of the dispensing arm where the tip contacts the plate. Dispensing is programmed to take place with the needle tip 80 micrometers from this reference Z-position which corresponds to the surface of the base-plate.
10. Position by eye the tip of the needle at the center of Well A1. Store in the controlling computer the reference X, Y coordinate of the dispensing arm where the tip is centered. This coordinate is used to define the center of the remaining wells on the plate.
11. Prime the syringe using the appropriate command on the robot computer to ensure that fresh mesophase fills the dispensing needle and that the first and subsequent wells receive their full complement of mesophase. Wipe off extruded mesophase on the needle tip with a tissue.
12. Start the Run Robot (X-AP) program to initiate a round of flushing steps where the precipitant dispensing tips on Arm 2 of the robot are washed and are prepared to aspirate solutions from the precipitant block.
13. Activate the dispense function on the robot to set in motion the sequential loading of wells with protein-laden mesophase (usually 50 nl) first, a column of eight wells at a time, followed by precipitant solutions (usually 800 nl). Between loading columns, the mesophase dispensing arm returns to a parking position where the end of the needle is placed in a moistened sponge to prevent the mesophase at the tip from drying out. The process of dispensing mesophase and precipitant is repeated 11 times across the plate until the plate is filled. The loading of a 96-well plate with mesophase and precipitant takes 5 min to complete.
14. As soon as the plate is filled and the robot arms have returned to their park positions, remove the plate from the deck of the robot and place it on a flat solid surface.
15. Immediately, seal the plate with a cover-glass to minimize time-dependent changes in well composition. It is important to ensure that all 96 wells are fully covered, that the cover-glass is aligned 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.
16. Inspect the plate to be sure the set up went as it should. What one is looking for is a ‘fried egg on a pan’ appearance in each well. The yolk of the fried egg corresponds to the mesophase. 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.
17. Place the plate in a temperature-controlled storage area for crystal growth.
18. If no more plates are to be set up, remove the precipitant block from the deck of the robot, seal it tightly and put it back into storage.
19. Remove the syringe containing the mesophase from the dispensing arm of the robot, dismantle it, and wash its parts including dispensing needle and ferrule, carefully with methanol and dry in preparation for next use.

3. In Meso Robot 2. Mosquito LCP

The Mosquito LCP robot (Figure 4) incorporates a similar mesophase dispenser to that used by Robot 1. In contrast to Robot 1, it dispenses precipitant by means of disposable tips. Programming software is used to set up the instrument for working with particular crystallization plates and precipitant blocks, and to dispense user-defined volumes of mesophase and precipitant in a specified sequence. Such details are provided by the manufacturer and will not be described here.

1. Prepare a Hamilton syringe, complete with needle and filled with protein-laden mesophase, as described above under Robot 1 and in JoVE article 1712¹.
2. Clamp the syringe in position on the dispensing arm of the robot.
3. Use the deck mounted laser sensor to establish the X and Y coordinates of the needle on the dispensing syringe. This is an important step required to ensure that the mesophase is dispensed accurately at the center of each well on the plate.
4. With the touch of a button on the control panel, prime the syringe to expel mesophase from the needle. Remove extruded mesophase from the needle tip with a tissue in preparation for using the robot to load wells.
5. Before placing the crystallization plate on the deck of the robot moisten the surface of the platform with a few drops of water to help hold the plate in place by capillarity.
6. Remove the protective cover from the spacer on the glass base-plate and place it on its platform firmly to ensure good adherence. Use the three fiducials on the platform to align the 3 corner wells thereby ensuring proper positioning of the wells on the plate with respect to the dispensing needle tip. The instrument is now commanded to enter a dispensing cycle.
7. The cycle begins with a second priming step where mesophase is dispensed onto a small glass plate immediately before moving on to load wells with fresh mesophase. The robot first loads with mesophase a vertical column of 8 wells on the plate and then uses the disposable tips to dispense precipitant solution on top of the mesophase bolus. The process is repeated 11 times along the length of the plate until all wells are filled. The entire process takes about 5 min to complete.
8. Remove the filled plate from the platform of the robot and seal it with a cover-glass as described above under Robot 1.
9. Place the plate, properly labeled, in a temperature controlled environment for crystal growth.

4. In Meso Robot 3. Gryphon LCP

The Gryphon LCP robot (Figure 5) incorporates a similar mesophase dispensing system to that described above for Robots 1 and 2. What distinguishes it from the other two is that all 96 precipitant solutions are dispensed simultaneously.

1. Prepare the protein-laden mesophase and load it into the dispensing syringe, as described above, under Robots 1 and 2, and in JoVE article 1712¹.
2. Clamp the syringe in position on the dispensing arm of the robot.
3. Place the precipitant solution block and crystallization plate on the deck of the robot.
4. Load the 96-tip head with precipitant solutions. A user-defined delay time is provided to facilitate aspirating viscous precipitants.
5. Command the instrument to enter a dispensing cycle. The cycle begins with the mesophase being dispensed sequentially into the 96 wells in a process that takes about 1 min to complete. The 96-tip head then dispenses all 96 precipitant solutions at once on top of the mesophase boluses in the crystallization wells. The entire plate loading process is finished in 2 min.
6. Remove the filled plate from the platform of the robot and seal it with a cover-glass as described above under Robot 1.
7. Place the plate, properly labeled, in a temperature controlled environment for crystal growth.

5. Representative Results

The purpose of this video article is to demonstrate how to set up automatically crystallization trials with a viscous, membrane protein-laden mesophase in glass sandwich 96-well crystallization plates. The emphasis is on automation through the use of specialized robots that require minimal manual intervention. Reproducibility and precision, in terms of volumes of mesophase and precipitant dispensed and the placement of both within the well, are expected when a robot is used. These are important from the point of view of effective screening of conditions that support crystallogenesis. They are also critical for the next step in the analysis pipeline which is to evaluate crystal growth often done with imaging equipment. Drop or bolus location is key to the imaging process, and irregular and improper placement of bolus in the well usually requires user intervention to correct for and takes time. For purposes of illustration, examples of properly and improperly loaded plates are shown in Figure 6.

Figure 1. The flowchart summarizes the steps involved in the production, harvesting and cryo-cooling of in meso-grown membrane protein crystals. Steps surrounded by the dashed red line are the focus of this JoVE article. Robots were used to dispense mesophase and precipitant solutions in Steps 29-32. From Reference 3. Click here to view larger figure.

Figure 2. A fully loaded and sealed 96-well glass sandwich crystallization plate. Each well contains 50 nl cubic phase and 1 μl precipitant solution. For clarity, the cubic phase has been stained with Sudan Red and the precipitant solution includes Methylene Blue. From Reference 5.

Figure 3. The in meso robot (Robot 1). The robot has two arms. Arm 1 dispenses the protein-laden lipidic mesophase. Arm 2 dispenses precipitant solutions. During filling, the 96-well plate remains fixed in position on the deck of the robot which itself is stationary. The two arms move to deliver mesophase and precipitant solutions. From Reference 3.

Figure 4. The Mosquito LCP robot (Robot 2). In contrast to Robot 1, the deck of the Mosquito LCP, upon which sit the 96-well plate and the precipitant solution block, moves horizontally during filling.

Figure 5. The Gryphon LCP robot (Robot 3). During a set up, the lipidic cubic phase dispenser moves to deliver protein-laden mesophase onto the 96-well plate. The deck then translates horizontally to position the plate under the precipitant solution tip head. All 96 precipitant solutions are dispensed simultaneously. Picture sourced from the manufacturer (http://www.artrobbins.com/) and labeled.

Figure 6. A fully loaded and sealed 96-well glass sandwich plate (A) and wells properly (B) and improperly (C, D) filled using the in meso robot (Robot 1). The bar code label on the plate in A is used to track the plate in the plate incubator/imaging device. In panels B-D, precipitant solution and mesophase are marked P and M, respectively. The expected ‘fried egg on a pan’ appearance of the well in B is characteristic of a properly loaded and sealed well. In C, the precipitant has been dispensed into the well improperly in an off-center position and has come in contact with the spacer. In C, it would appear too that the volume of mesophase is less than it should be. An example of incomplete loading with precipitant solution is shown in D. For scale, the diameter of all wells is 6 mm.