Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

Hello, and welcome to this JoVE"video. We want to introduce you to the setup that we've got here for rapid grid making, a time-resolved study that we're developing over in Leeds. Be aware this isn't a standard system, but we hope that by giving you a detailed account of all the things and the parameters that we do, you'll be able to develop similar systems in your own laboratories.

A detailed description of the device is given in the Richmond protocol. Here, we're going to give a step-by-step guide for fast grid preparation and time-resolved EM.In our experience, protein concentration should be two milligrams per milliliter or higher. The protocol requires a minimum volume of about 50 microliters.

Turn on the device. Then turn on the control PC and start the control software. Initialize all syringe pumps by pressing the initialize button on each syringe pump.

Turn on the potentiometer power supply, set it to nine volts, and start the oscilloscope control software. Pressurized nitrogen gas is needed to generate the spray. Open the nitrogen cylinder and set the pressure to 2 bar.

Ensure all syringe pump valves are in load position. To do this, switch all valves to dispense in the control software and then back to load. Set all syringes to zero.

Any air bubbles present in the system should be removed at this stage. To do this the syringes may have to be unscrewed, bubbles removed manually, and the syringes mounted again when containing no more bubbles. The liquid handling system is usually stored in water.

Before loading the sample, the system is equilibrated with buffer. This is done by washing the tubing with an excess of buffer. Place a tube containing at least 200 microliters of buffer onto syringe one.

The top of the tube must be pierced to attach it. Aspirate a suitable amount of buffer, between 50 and a hundred microliters with syringe one using the control software. Switch valve one to dispense.

Dispense all of the liquid in syringe one. Return valve one to the load position, and press the initialize button on syringe one to prepare the system for the next wash cycle. These steps are usually repeated three times to ensure thorough washing of the tubing.

Next, the spray nozzle is positioned. Place an EM grid in the plunging arm. Position the grid in front of the spray nozzle.

Dispense buffer using the control software. If nozzle and grid are aligned, liquid should accumulate after spraying for an extended period of time. If necessary, adjust the nozzle position and then check again where the liquid accumulates on the grid.

Next, adjust the position of the ethane cup. The tip of the tweezers should reach approximately to the center of the ethane cup. Perform a test run to ensure all settings are correct.

Close the humidity chamber. Make sure the path of the plunger is clear. Aspirate the volume of buffer required for a single run into syringe one.

Switch valve one to dispense. Start the run by pressing run'in the control software. Check that syringe pump one is moving, two seconds in this case, and the grid is plunged after an appropriate delay, 1.5 seconds here.

When the run is finished, set the pressure on the plunger arm to the desired value. Press OK'in the control software to release the pressure from the plunging arm. Now the ethane cup is cool and filled with liquid ethane.

Glow-discharge the grids before use. A typical glow-discharge is 90 seconds at 10 milliamps in 0.1 millibar air. The tubing is then equilibrated with sample.

If the available sample volume is low, the tubing may be equilibrated with only one dead-volume. Aspirate sample. Typically the dead volume is around 30 microliters.

Switch valve one to dispense. Dispense the sample through tubing and nozzle. Then aspirate the amount of sample required for a single run.

Switch valve one to dispense. Check that the relative humidity has reached the desired value. We typically prepare grids at 60%or higher.

This can take a few minutes. Place the tweezers holding a glow-discharged grid in the plunging arm. Place the potentiometer slide in the start position, ready for speed measurement.

Set the trigger for the speed measurement in the oscilloscope software. Place the liquid ethane cup, and close to humidity chamber. Make sure the path of the plunger is clear.

Start the run in the control software. When the run is finished, press OK'in the control software to release pressure from the plunging arm. Open the humidity chamber, loosen the connection between plunging arm and tweezers.

Move up the plunging arm while keeping the grid in liquid ethane. Then transfer the grids to liquid nitrogen and into storage. Save the oscilloscope measurement.

Manually reset the position of the potentiometer slider and plunger. Repeat the protocol to prepare replicate grids. Time-resolved experiments are done in a similar way.

Higher stock concentrations are needed for time-resolved experiments because of dilution in the mixing step. For a time-resolved experiment, use all three syringes. Tubing is attached to the syringe pumps and to the spray nozzle.

The microfluidic spray nozzle used here also contains a mixing element. Note that longer tubes will give a larger dead-volume and washing equilibration steps require more buffer and sample. Equilibrate all syringes with buffer and sample separately.

Typically, syringe one is used for sample A'and syringes two and three are used for sample B'After the tubing is equilibrated, load sample A into syringe one and sample B into syringes two and three. Then, switch valves one to three to dispense. Start the run in the control software.

Note that a different run script is needed for a time-resolved experiment. Different time delays can be achieved in two ways. By adjusting the plunger speed, the time delay from mixing to freezing can be changed.

A faster plunge speed will lead to a shorter time delay. Alternatively, the spray ethane distance is changed by adjusting the vertical position of the spray nozzle. Increasing the distance will result in a longer time delay.

In the electron microscope at low magnification, a typical grid looks like this. Areas of thin ice, as indicated, are suitable for data acquisition. At higher magnifications, particles should be clearly visible.

With a test specimen such as apoferritin, a relatively small data set from a single grid is sufficient for a reconstruction at three to four angstrom resolution. For time-resolved experiments, we collect data sets from different time points or grids. It's useful to combine the data for 3D classification and then trace particles back to their time points.

This way the data can provide structural information and information on the reaction kinetics. In conclusion, we hope you enjoyed the video. We hope that with the detailed descriptions in the JoVE paper along with the video, you'll be able to conduct your own experiments via rapid grid making, which has been shown to alleviate some of the problems from mutation or to help with interactions at the air-water interface, all you need for conducting your own time-resolved studies to try and trap some of those non equilibrium states.

So, happy grid making.