Automated Protocols for Macromolecular Crystallization at the MRC Laboratory of Molecular Biology

The overall goal of this procedure is to produce high-quality crystals from initial crystallization screening conditions. The crystals can be used to study the structures of proteins, DNA, RNA, and their complexes. Something about macromolecular structures has become increasingly challenging.

And providing a broad variety of initial conditions increases the yield of useful crystals, while reducing the need for sample and crystal optimization. The two fully automated systems, the the LMB, facilitate the preparation of up to 1, 920 vapor diffusion experiments in nanoliter droplets. For brevity, it is assumed that users are already familiar with the basic functions and maintenance of the nanoliter dispenser, the inkjet printer, and the adhesive plate sealer.

Prior to performing the procedure, ensure that the system's robots are initialized and their controlling software is open. Turn on the chiller of the tube cooling carrier, about 30 minutes before the main program will be started. Place a test plate on the motorized SBS carrier of the plate sealer.

Run the plate sealer and check that the film is properly applied. Repeat this test twice more to verify that the plate sealer is ready. Then add 20 liters of deionized water to the main container of the liquid handler.

Disconnect the coupling inserts form the smaller container of 20%ethanol rinsing solution, and connect the inserts to the main container. Enter the appropriate screen name on the inkjet printer touchscreen. Then open and close the carousel door to trigger the carousel rotation.

Open the door when the first stack presents itself. Fully load the first stack with 22 crystallization plates, each with column one facing out. Fully load the next two stacks in the same way.

Load the remaining six plates in the highest positions of the fourth stack, then close the carousel door. Verify that the chiller display indicates 14 degrees Celsius. Gently and repeatedly invert the selected screening kits for one minute.

Then, open the front panel of the liquid handler. Open the tubes and place them in the cooling carrier in the standard 96-well layout. Upon placing each tube in the carrier, place its lid on the tray in the same 96-well layout.

When using System 1, the label leads are placed on the tray in the 96-well layout to enable cross-checking that the tubes are arranged correctly. Cross-check the tube positions and then ensure that all tubes are level and settled in the carrier. Then close the front panel.

In the Startup window of the liquid handler software, select Run Maintenance and open the Flushing program. Run the program to flush the 20%ethanol from the system and to wash the outsides of the liquid dispensing tips. When flushing is complete, click Cancel to return to Startup.

Select Run An Existing Process and click Start Your Selection. Open the MRC Kit dispensing program. Fill in 18"for Instances in the Configuration screen and run the program, Monitor the system as the first four plates are labeled, filled, and sealed.

Do not leave the system unattended until the preparation of the first four plates is complete. Once all 72 plates have been prepared and placed back in the carousel, switch the coupling inserts from the deionized water container to the 20%ethanol container. Run the startup flush program using the same process as before.

Turn off the chiller and discard the empty screening kit tubes. Carefully remove the 72 prepared plates from the carousel. Discard incorrectly filled or poorly sealed plates.

Store the ready-to-use plates at 10 degrees Celsius. First ensure that the system's robots are on, the nanoliter dispenser is initialized with its software open, and the liquid handler's method manager is open. Turn on the microtube cooling carrier about 15 minutes before the main program will be started.

Place a test plate on the motorized SBS carrier of the plate sealer. Run the plate sealer and verify that the plate is properly sealed. Test the plate sealer three times.

Then run the nanoliter dispenser to set 100 nanoliter droplets of test solution in a test plate. Shown here is a light micrograph of a 100 nanoliter droplet, produced by the nanoliter dispenser, with the test solution made with red dye. Close the nanoliter dispenser software and remove the strip holder block from the deck.

Next, set in a custom-designed plate holder, a pre-filled 96-well crystallization plate with the highest relative quantity of volatile reagents in its conditions. Remove the adhesive film from the plate. Open the liquid handler front panel, and place the unsealed plate on the deck at the back of the first sliding carrier.

Cover the plate with an SBS aluminum lid. Settle the lid towards the rear left corner of the carrier by applying gentle pressure to the opposite corner of the lid. When using System 2, screening kits with significant amounts of volatile reagents should be processed first.

This avoids condensation forming on the SBS leads, which could effect automated handing of the leads or plate sealing. Unseal, load into the sliding carriers, and cover the remaining 19 plates in the same way, working from most to least volatile conditions. Once the microtube cooling carrier is at 4 degrees Celsius, as indicated by a green light, remove its cover.

Cut off the lid of a microtube containing at least 440 microliters of a protein sample. Ensure that the sample has no foam above the meniscus, as this will interfere with the liquid detection system. Place the tube in Position 1 of the microtube cooling carrier.

Place a PCR plate on the liquid handler deck in front of the plate moving adapter carrier. Then close the front panel. In the liquid handler method management interface, select Setup Plates.

Monitor the initialization of both systems and fill in the run parameters, including the plate type and the number of drops for each condition, the drop size, the position of the first plate to be processed, and the number of plates. Click No when asked if you would like help loading the deck. Once you've checked that there are no empty tip racks, click OK.Once you've checked that there are enough tips for the run, click OK.When necessary, update the tip management system and click OK.Ensure that the nanoliter dispenser deck is clear of the strip holder block, and that the carrier is at the back of the 50 microliter tip stacks, are clear of the aluminum SBS lids.

Double-check that all the other required components are ready and then start the process. Monitor the system as the nanoliter dispenser sets the drops in the first plate, and the plate sealer subsequently seals the plate. Do not leave the system unattended until the preparation of the first plate is complete.

Once all 20 plates have been prepared with crystallization droplets and automatically return to the sliding carriers, open the front panel and gently remove the plates. Check that the plates are correctly sealed before storing them for crystallization. Clean the SBS lids with the 20%ethanol solution before stacking them on the left-hand side of the liquid handler for storage.

Discard the PCR plate and the microtube. Turn off the microtube cooling carrier and wipe away the condensation. Leave a paper towel on top of the cooler surface to absorb further condensation.

Then replace the carrier cover and close the front panel. OmpF and MreB were initially screened using these fully automated protocols. OmpF crystals of sufficient quality for X-ray diffraction were found in well A4 of the LMB 7 plate.

Diffraction quality crystals of MreB were found in well D12 of the LMB 20 plate. Storing pre-fuel plates at 10 degrees Celsius is a compromise to avoid freezing and storage at 4 degrees, which may cause deterioration of crystallization conditions and condensation-related issues. When stored this way, plates may be used within four to five months.

Between 4 and 8, 000 LMB plates are produced yearly with System 1 and are later used for initial screening. The protocols should be optimized differently when the expected turnover is much lower. Our entire set of pre-fuel plates is normally used as a large initial screen for a novel sample.

However, fewer plates may be selected to much specific sample requirements. After preparing the droplets, plates are stored on low-vibration shelves with the temperature tightly controlled at 4 or 18 degrees Celsius. Experiments are assessed with scroll light source microscopes.

After carefully investigating the nature of the crystals, traits and similarities across conditions can be analyzed with web-based tools such as the LMB or C6 Screen databases. Subsequent optimization of crystallization conditions may still be necessary. Additional automated protocols, with the 4-corner methods and additive screening are described in our manuscript.