Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

Using the Spotiton robotic system, one can mix and vitrify a protein of interest with an interacting partner on an electron microscope grid in as fast as 90 milliseconds. This protocol allows the capture of intermediate protein confirmations that are too transient to be captured by standard grid preparation techniques. Time resolved Spotiton can inform sub-second biological or biochemical systems such as membrane channel activation, DNA or RNA synthesis, or early interaction of a drug or antibody with its target protein.

The user must concurrently manage several components that directly impact the quality of a grid. It is important to understand the system before using and have patience when preparing grids. To begin, open the main valve on the nitrogen supply tank and ensure the system reservoir is filled with de-gassed ultrapure water.

Turn on the computer and the Spotiton system at the multi-outlet power strip. Click on the desktop icon to open the user interface of the Spotiton software. In the Tools menu, select Initialize Stages to initialize and home the three axis robots and rotating dispenser head assembly, ensuring the dispenser tips are pointing down prior to initialization and homing.

On the main menu, click on Go to Safe Position to send the robots to the safe position. On the Aspirate tab, select Prime to flush the dispenser heads multiple times with water from the reservoir. Continue until two uninterrupted streams of water can be seen emerging from the tips.

On the Inspect tab, send tip one to the inspection camera and test fire water, adjusting amplitude until discrete droplets are produced. Press the Record button in the upper camera monitor to record a video of tip one firing. Send tip two to the inspection camera.

Play back the video of tip one firing in the right side monitor at the same time tip two is fired in the upper camera monitor, matching the pattern of droplet production from the two dispensers. Remove the tweezers from the mount on the grid robot using the supplied Allen key. On a nearby benchtop, position a test grid nanowire side up on the edge of the grid block.

Carefully grab the rim of the grid and position it correctly in the tweezers. Then remount the tweezers. On the Cryo tab, click on Tip to Camera to move tip one into the field of view of the upper plunge path camera, ensuring that Live is selected in the upper camera monitor.

Turn on and adjust the upper camera light. Position tip one visible in the upper camera monitor, by clicking the mouse within the monitor. Click on Grid to Camera to position the grid in front of the upper camera, then adjust tip one position again if needed.

On the Cryo tab, ensure Vitrified Grid is not selected, click on Queue Target, then on Plunge. Evaluate the upper and lower images to confirm the dispensers are functioning normally. Dilute two samples to the desired concentrations with an appropriate buffer, ideally using the same for both, and fill the cryogen bowl with liquid nitrogen.

Plasma clean three to four nanowire grids using five watts hydrogen and oxygen, and 1.5 minutes as the starting point. Plug in the nebulizer and observe the vapor exit the central port of the nebulizer cap. Observe the Live Humidity Monitor in the main window or open the Ambient Humidity Tracker under Reports and Ambient.

Check humidity levels in the chamber and shroud zones. Add five microliters of each sample into the sample cups. Load the sample cups into the holding tray with a sample for tip one on the left and for tip two on the right.

Then push the tray back into the machine until it seats. On the Aspirate tab, select three microliters for the volume to be aspirated by each tip. Ensure the sample tray has been seated securely, click Aspirate, and observe the pipette stage move the dispenser heads into the sample cups.

Verify successful aspiration of both samples by removing the sample cups and observing a drop in the liquid levels. On the Inspect tab, send each tip to the inspection camera to confirm unobstructed dispensing. Adjust the amplitude as needed to match droplet formation from each tip.

Load a freshly plasma cleaned grid into the tweezers, but do not mount the tweezers yet. Ensure that the humidity level is elevated to approximately 90 to 95%Fill the ethane cup and perform a final test fire of both tips in front of the inspection camera, confirming no obstruction. On the Cryo tab, click on Tip to Camera.

Check ethane cup. If ethane ice has formed, melt as needed with additional ethane gas. Mount the tweezers with the grid onto the grid stage.

On the Cryo tab, click on Grid to Camera, ensuring tip one is positioned correctly in the upper camera monitor. Click on Vitrified Grid, Queue Target, then Plunge. Click on OK when prompted to command the grid robot to hop the grid from ethane into liquid nitrogen and release it onto the submerged shelf.

Examine images of the grid to decide if it should be kept or discarded. If keeping the grid, pre-cool fine-tipped forceps, gently grasp the grid by the edge, and place it into a grid box slot, starting with the first slot on the left of the notch and going clockwise. Use the Experiment Viewer to review and compare the grid images from the upper and lower cameras along with the machine settings and humidity measurements at the time of the plunge by selecting Reports and Experiment.

Images of grids prepared during a single time-resolved Spotiton session by mixing RNA polymerase in a 105 base pair DNA oligomer carrying a promoter sequence for 150 milliseconds prior to vitrification are shown here. Of the six grids, only one shows suboptimal wicking. The pattern of ice deposition on a vitrified grid matches closely with the pattern of deposited liquid seen in the upper camera image.

Effective wicking of the mixed samples occurs along the nanowire covered grid bars where the sample rarely overflows into squares adjacent to those in which it landed. In ice-filled squares, the ice is typically thickest within holes at the center of the square and becomes thinner in holes closer to the grid bars. Holes immediately adjacent to the grid bars are often empty, due to proximity to the nanowires.

Proper preparation and handling of the nanowire grids will ensure good ice thickness on mixed sample grids. The Spotiton system also allows the user to deposit the two samples separately on a single grid, enabling the collection of an unmixed control during the same grid maintenance session. Spotiton has enabled the capture of the first intermediates in bacterial gene expression in real time.

Because they form on a sub-second timescale, their structures have been unknown until now.