Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

The goal of the following experiment is to be able to image macromolecules in their frozen hydrated state with the transmission electron microscope. This is achieved by flash plunging the grid with the sample onto the cryogen to get a thin vitrified specimen. As a second step, the sample is exposed to the electron beam, which will form a projection image of the macro molecules.

Next, the images are processed on the computer in order to obtain the 3D reconstruction of the macro molecule. The results show a highly detailed 3D structure of the macro molecule, its confirmation, and the 3D location of its molecular partners. There is a main advantage of this technique, of other methods of sample preparation for electromicroscopy.

In cryo, the image is directly formed by the electrons within the macromolecule. This reflects more faithfully, its near Native structure. A visual demonstration of this method is critical.

The cryo grid preparation and is transferred to the electron microscope are difficult to learn because of the additional complication when working at cryogenic temperatures, The warmup cycle is used at the beginning of the pumping sequence and after a cryo EM session. To begin this procedure, insert the cryo holder into one of the pumping ports of the pumping station. Start the warmup cycle option.

In the cold stage controller, open the butterfly valve V two, and when the vacuum in the turbo pumping station stabilizes and reads 10 to the minus three tour, or better open the butterfly valve V one. Once the temperature in the holder is stable above room temperature and the warmup cycle is complete. Lows V one and stop the cycle.

Next, start the ze light cycle option and select the necessary time to attain a good vacuum in the range of one to two times 10 to the minus four tor. When the vacuum reaches 10 to the minus three to range open the DU evacuation valve B three and the valve on the cryo holder. When the cycle is complete, rose the valves in the opposite order in which they were opened.

Using a pair of tweezers, leave a piece of MICA to expose two new surfaces. Place the new exposed mica surfaces face up on a white piece of paper in a Petri dish. After placing the Petri dish in the be jar of a carbon evaporator, perform the pump down sequence until the vacuum is below 10 to the minus six tor coat the mica with a thin layer of carbon.

Then transfer the thin carbon to the grid. That a 0.5 by one centimeter piece of coated mica and float the carbon off onto the flat surface of filtered deionized water. Using the tweezers, put the holy carbon side of the grid in contact with the upper surface of the floating carbon layer and pick it up.

Place the grid with the carbon side facing up on a 7.5 by 2.5 centimeter glass slide covered with power film. Then place the glass slide into the chamber of the glow discharge equipment and close the chamber. Pump the bell jar and glow.

Discharge for 20 seconds at 25 milliamps and seven times 10 to the minus two millibars. At this point, turn on the plunge freezing apparatus. Pull the Ethan container down by filling the reservoir with liquid nitrogen.

Once the liquid nitrogen stops boiling, place the tip of the tube coming from the Ethan tank in the inner chamber and open the valve very slowly. Liquefy the ethane in the inner chamber and fill it to one millimeter from the top. Turn on the humidity.

Next, load a grid onto tweezers and load three to five microliters of sample onto the carbon surface of the holy grid. After waiting for the sample to absorb to the grid, perform blotting To achieve a thin liquid layer blush, freeze the grid by plunging it into the liquid ethane. Carefully remove the tweezers grid assembly from the liquid ethane and transfer it to the outer chamber containing liquid nitrogen.

Then transfer the grid to a small grid box immersed in the liquid nitrogen. At this point, insert the cryo holder into the cryo transfer workstation. Fill the doer of the cryo holder and the workstation vessel with liquid nitrogen.

Quickly transfer the cryo grid box containing the frozen hydrated grid to the liquid nitrogen in the workstation. After opening the cryo grid box, take the frozen hydrated grid and place it into the sample holder slot. Next, place the clip ring on top of the grid and gently press it with the blunt end of the clip ring tool and until it is firmly seated in place.

Then close the cryo shutter. Top off the TEM anti contaminate with liquid nitrogen. Re tilt the microscope stage to minus 60 degrees.

Following this, begin the PrepU airlock cycle on the microscope. Once the pump airlock sequence has finished, insert the choir holder into the airlock and connect the cold stage control cord to the holder to monitor the temperature. After the red light is extinguished, rotate the holder to the zero degree position for its insertion into the T EM'S high vacuum column.

Retract the cryo shutter and open the column valves. Activate the low dose program in search mode. Center the stage and set the centric height using the alpha wobbler to rock the stage plus or minus 15 degrees, select a suitable hole and place it in the center of the field with the XY stage controller.

Switch to focus mode and focus the image. Then under focus the image between 0.5 and 4.5 microns. Switch to exposure mode and record the image.

Finally, return to search mode to select the next hole and repeat the sequence. Microscopic images obtained by the methods of negative staining and cryo EM for four representative specimens with different structural characteristics are compared here negatively stained adeno-associated virus capsids reveal virus-like particles and cryo EM shows they are empty. Images of tobacco mosaic virus show helical rods with the central channel filled with stain in the negative staining and empty in the cryo EM images.

KLH assembles in characteristic barrels showing six striations in both negative staining and cryo EM images. Rectangular side views and circular and on views reveal an empty in a structure by cryo EM in the odine receptor. Intricate surface features such as a central cross and a central depression are manifested as stain accumulation by negative staining and as lower density regions by cryo em.

Typical examples of cryo EM artifacts are crystalline ice, ice contamination, astigmatism, and web-like structures. Single particle analysis and 3D reconstruction of frozen hydrated ine receptor one reveal its fourfold symmetric structure. The cytoplasmic domain forms a square prism and contains several reproducible gular domains forming a scaffold like structure.

At 10 angstrom resolution, it is possible to visualize alpha HELOCs 3D. Difference mapping can reveal the position of important ligands. Confirmational changes provide a dynamic view of the macro molecule.

After watching this video, you should have a good understanding of how to prepare high quality cryo EM samples. Cryo EM combined with image processing, has enabled researchers to explore the structure subunit organization and conformational dynamics of macromolecular machines.