Remote Access at Non-Cryogenic Conditions and Controlled Humidity

2. Disposable Supplies for Crystal Growth inside Plates - Supplies include silicone cup liners (CP-111-093) and absorbent foam inserts (CP-111-094) that hold crystallization mother liquor solutions and 1” crystal clear tape (CP-111-092) to seal the plates (Fig.4). Figure 5 shows the absorbent foam inserted into a cap which is then inserted in a plate well.

3. Blue Thermal Shipping Container - the container holds up to six plates in foam holders for safe shipping to SSRL (Fig. 6). The thermal shipper contains a phase-changing liquid that can maintain samples at 20 ^ᵒC (+/- 5 ^ᵒC) for up to 7 days during shipment. A 5 ^ᵒC thermal shipper is also available online through Crystal Positioning Systems (CP-111-105-20 or CP-111-105-05). The shipper is also useful to ship standard SDC format microplates and comes with gel cooling blocks to further stabilize the inside temperature.

4. SSRL Grid Mounts - the grid mounts are useful for both crystal growth and diffraction data collection and are commercially available at MiTeGen. There are two types of grid mounts available online: 800 micron windowed grids and square windowed grids. For more information about Grids, see this open-access Article.

Before each use, the plate should be visually inspected to be sure it is free of dirt and debris (Fig. 7a). Also, inspect the magnetic rings for any signs of corrosion (Fig. 7b). Corrosion can affect the ability of the robot to remove and replace pins. To prevent corrosion of magnets, remove disposable cups containing solutions when plates are not in use. Plates should be stored dry.

Note: when the plates are not being used, remove all tape, dry the plates and store then in a dry dark area. Plates left in sunlight or under fluorescent lights may turn yellow with age. The yellow plates are still OK to use, but less light will transmit through the plate when viewing the samples through a microscope.

The SSRL in-situ crystallization plate is compatible with a variety of sample holders (meshes, chips, capillaries, grids, etc.) affixed to magnetic sample pins (Fig. 8). It is important to choose magnetic bases that are compatible with the Stanford Automated Mounter robot (SAM). Detailed instructions about the allowed sample bases for use with the SAM robot can be found in the SAM Manual, including a size chart for sample pin length and details about sample pin vendors and part numbers.

Note: The SAM system supports only Hampton-style CrystalCap Copper Magnetic pins or CrystalCap Magnetic pins.

SPINE pin bases are not allowed because they will cause damage to the SAM robotic system.

Critical: It is imperative to use the proper length and width samples to ensure reliable operation with the SAM robot. Otherwise, the robot grippers may crush and damage samples.

Fig. 9 shows a capillary that is too long, placed inside SAM robot grippers (i.e. cryo-tongs). When the robot grippers close, this capillary will be crushed (but it would be OK if the capillary was 1 mm shorter).

Note: To make testing the sample pin size easy, a pin tester has been incorporated into every SSRL In-situ Plate.

The pin tester consists of a cavity and a side pocket (Fig. 10a) where sample pins can be tested. If the pin fits inside the cavity with the back of the pin flush to the surface of the plate (not sticking out past the surface of the plate material), then it should fit well inside the SAM robot cryo-tong/gripper. If the sample pin does not properly fit inside the cavity, a side cutout (Fig. 10b and 10c) helps with inspection of the assembly to locate the parts that are too large.

Bottom-line, if the sample pin assembly has a solid base and properly fits inside the SSRL In-situ Plate testing ports, it can be mounted by the SAM robot for remote data collection. This provides flexibility in the types of samples that users can ship at ambient temperatures and the types of experiments that can be supported on the SSRL beamlines.

Remote room temperature and multi-temperature experiments are possible by shipping samples in appropriately sized sealed capillaries or microfluidic chips (see above for Proper Dimensions). For example, Fig. 11 shows crystals inside a sealed thin capillary that can be mounted remotely from plates. See the following section for Instructions on the Use of MiTeGen Sleeves or Capillaries.

The Oxford Cryo-cooler system used at most SSRL beamlines can operate at a temperature range between 100 K and 370 K. Normal operation is at 100 K with no user adjustments enabled. However, if you send sealed samples in the SSRL In-situ Plates, you can ask your user-support scientist to enable remote adjustment of the temperature from the Blu-ice control software Sample Tab (Fig. 12).

The green area displays the current sample temperature. To change the set point temperature, type a value in the entry box (which will be in red text) then click “Move” to change in set point (the “set point” text will then turn black).

A popular method to ship samples at room temperature is in MiTeGen sleeves or capillaries.

Note: If sleeves or capillaries are used with plates, the instructions are different from the standard methods.

Instructions for using sleeves or capillaries:

1. It is important to cut sleeves or capillaries to a short length that fits in the Plate Tester.

Critical: If the capillaries or sample holders in sleeves are too long, they will be crushed by the sample mounting robot (see Fig. 9 above)

2. Epoxy the MiTeGen sleeves or capillaries in place.

Critical: Do not use grease or wax of any kind. Grease and wax will interfere with robot operation.

3. Add approximately 4 µl of crystal mother liquor solution in the sleeve before sealing it. Alternatively, a drop of mother liquor with ~1% of agarose added may be used. The addition of agarose prevents the drop from moving and recent testing has indicated it better stabilizes the crystal environment compared to mother liquor alone.

Note: To prevent crystal slippage when using sleeves, a key step is using a loop that is appropriately sized to hold the crystal so it doesn't have anywhere to go. The use of MiTeGen "Microgrippers" or meshes with excess solution wicked away can be helpful as well.

4. Properly insert the cups and absorbent foam inserts into the bottom of the plate chambers (as described in Growing or Transferring Crystals Inside Plates) and fill with water or diluted mother liquor solution. This creates a secondary humid environment surrounding the polymer capillaries or sleeves.

Note: While MiTeGen sleeves and most polymer capillaries are not watertight, glass or quartz capillaries properly sealed with epoxy are watertight and do not require any additional solutions to be added in the plate chamber.

5. After water or mother liquor solution is added to the chamber cups in step 4, seal the chamber on the top and side with 1 inch crystal clear tape (as described in Growing or Transferring Crystals Inside Plates).

Multiple crystal types have been shipped in plates using MiTeGen sleeves and glass capillaries for remote collection, often producing diffraction data between 1.5 and 1 Å resolution.

• For experiments under controlled humidity conditions, crystals may be grown in the SSRL In-situ Plates or bare crystals can be transferred into properly charged plates, following the instructions outlined the next sections of this document.
• To prevent crystals from moving around during data collection, we recommend growing or mounting crystals on meshes or grids that facilitate wicking away excess solution before shipping. For example, the images shown in Fig. 13 are of crystals grown and shipped from California to New York on windowed grids inside plates. Images of crystals were recorded in each location. Before shipping, the grids were removed to wick away excess liquid from around the crystals (using a piece of filter paper), and then returned to the plates.

• Crystals mounted onto the beamline goniometer are maintained at a set humidity level using an Arinax humidity control nozzle (Fig. 14A). The nozzle directs a stream of humid gas onto your crystal at a user specified relative humidity setting.

Important: Schedule with user-support a week in advance to ensure the Arinax humidity nozzle is in place for your experiment.

• Upon request, your support staff can also set the humidity level inside the robot dispensing box that is used to store your in-situ plates within the robot dispensing box (Fig. 14B)

• If you do not know the starting humidity level associated with your sample crystallization conditions, consult the Blu-Ice Sample Tab Manual for Determining a Relative Humidity Starting Point.
• The relative humidity level of the gas stream at the sample position can be set from the Blu-Ice control software Sample Tab view (Fig. 15). If you cannot see the humidity control options, ask your user-support scientist to enable humidity control for your experiment. The green area displays the current relative humidity. To change the set point, type a value in the entry box (which will be in red text) then click Move to change the set point (the new Set Point text will then turn black).

• Controlled dehydration experiments can be done remotely.

Recommendation: Although instructions are online, it is recommended to work closely with your assigned beam time support staff during your first controlled dehydration experiment.

The SSRL In-situ Plate can be used to grow crystals by hanging or sitting drop vapor diffusion on Grids or other substrates affixed to magnetic bases. Alternatively, crystals grown elsewhere can be transferred onto substrates stored in charged plates.

Note: Plates can be reused for multiple crystallization trials and trips to the synchrotron.

The steps below cover both in-situ growth and sample transfer scenarios.

1. Install silicone cup: To make cleaning the plates easy, the wells that hold mother liquor solutions are lined with disposable silicone cups (Fig. 16).

Following the instructions below, carefully install the cups to avoid problems with solutions spilling or collisions with the SAM robot:

• Using tweezers or a gloved finger, insert the silicone cups into the plate wells (see Fig. 17).
• Gently press the rim of the cup to ensure it is secured. You can feel the cup snap into place. If properly installed, the cup will remain in place when shaking the plate upside down.

Critical: If the cup is not properly inserted, it may interfere with insertion of the magnetic pin base.

2. Add absorbent foam insert and crystallization precipitant solution: This will avoid crystal de-hydration and mother liquor spillage during transport.

• Using tweezers insert the absorbent foam into the silicone cup (Fig. 18). It should easily drop into place.

• Use a pipet to fill the cup with 300 ul of the crystallization solution, soaking the foam insert inside with liquid.

Note: An option to ensure the foam is fully saturated, is to add excess liquid into the cup and then after a minute remove any excess liquid above the foam that has not been absorbed with a pipette.

Note: Adding 0.6% agarose to the well solution is an alternative approach to avoid spilling, if the absorbent foam inserts are not available. A video is available for detailed instructions on this process.

3. Seal the plate chambers: Use the 1” crystal clear tape provided in the kit. Seal both the top and sides of the wells, being sure to fully cover the openings and to smooth out any air bubbles to ensure a proper seal.

• Alternatively, the top of the plate may be sealed with Qaigen-plate screw caps.

• Avoid putting any clear tape across the top middle of the plate – this channel is used with a spring-loaded roller ball to hold the plate inside the shelf (Fig. 19).

4. Equilibrate plates and fill with protein sample: After sealing each chamber with tape, let the plate equilibrate for at least 30 minutes.

5. For crystal growth in plates: After 30 minutes, pipette protein/precipitant solution drops on a substrate, such a Grid or mesh affixed to a magnetic base.

• Quickly open the tape on the side of a port, transfer a magnetic bases with protein/precipitant drops inside.

• Carefully reseal the chamber.

• Use a dissecting microscope to view the plates periodically and monitor crystal growth on the substrate.

6. To transfer bare crystals grown elsewhere into plates: Carefully pickup crystals and place onto a mesh or grid affixed to a magnetic mount.

• Quickly open the tape on the side of a port and transfer the magnetic base with crystals inside. Carefully reseal the chamber with tape.

• Transferred crystals can survive in the plate for a week or longer.

7. Seals the plate: After all ports of one side of a plate is filled, to ensure a good seal, a single strip of tape should be affixed over the sample pins bases on that side (Fig. 20). The top should also be sealed with tape or Qiagen screw caps.

Up to 6 SSRL In-situ Plates can be safely transported in one Blue Thermal Shipping Container. The thermal shipper consists of many layers of material optimized for maintaining a constant temperature and for reducing physical shock to samples inside (Fig. 21).

The thermal shipper will maintain a temperature of between 15 and 25 ^ᵒC for 7 days. Additional gel packs within the shipper will further stabilize the temperature.

Follow these steps to ship plates to SSRL in the thermal shipper.

1. Complete the online Shipping Form.

The form will produce a shipping label to tape on the outside of your shipping box and a required return shipping form that should go inside the shipping box just under the polyurethane lid.

2. The thermal shipper includes removable chambers that contain a phase-changing liquid. Before shipping, these should be removed and equilibrated at 21 ^ᵒC in an incubator for 48 hours. Alternatively, the entire thermal shipper box can be stored in the incubator for 48 hours with the top lid open.

Caution: Make sure not to pierce the aluminum covers of the liquid filled chambers.

3. The thermal shipper has a black foam insert with cut-outs that snugly hold the plates. Place the thin layer of 1/16” foam sheet between the edge of the plates and the hard plastic of the container walls.

4. Underneath each plate is a removable foam block that can be replaced with two gel packs included with the thermal shipper kit (Fig. 22). If less than six plates are shipped, the extra space can be filled with larger gel packs that also included.

5. Three layers of foam inserts, plates and gel packs are stacked inside the thermal shipper.

6. Remember to insert the return shipping paperwork before adding the polyurethane lid and closing the blue corrugated outer box. Tape the outer box shut and tape the shipping label to the outside.

A specialized enclosed shelf can hold up to 5 plates (Fig. 24A) that is in reach of the SAM robot at the beamline. The shelf is enclosed inside a Plexiglas box (Fig 24B). The environment in this box can be maintained at a specified temperature and humidity.

A simple interface has been incorporated into the Blu-Ice Sample Tab to mount samples from the SSRL In-situ Plates located in the shelf (Fig. 25). Each position of the shelf is labeled A-E in the software with the top shelf labeled as “A”. Each plate holds ten samples – numbered 1-10 in the software interface.

Figure 25. Blu-Ice tab Sample Tab for mounting samples from SSRL In-situ Plates.

To mount a sample, select Plates from the sub-tab of this interface. Click on the desired sample pins to mount (samples that are OK to mount are highlighted in green) and then click the Mount Button. Samples can only be mounted from plates that have been assigned to your account.