The functions of proteins are temperature dependent and are optimal at the temperature. This protocol provides a way to solve protein structures at higher temperatures. This approach can provide functionally relevant structures and enhance the understanding of the temperature dependence of protein structures.
Demonstrating the procedure will be Yuan-Chih Chang, Manager of the Academia Sinica Cryo EM facility. Begin by making a one centimeter hole in a 50 millimeter centrifuge tube on its closed end. Place the tube in the vitrification apparatus chamber at the ultrasonic water outlet.
Set up the vitrification apparatus temperature to the specified temperature, and allow the vitrification apparatus chamber to reach 55 degrees Celsius and 100%relative humidity. Let it stand for at least half an hour to stabilize the conditions before starting the experiment. Place the water bath on a hot plate and set the hot plate to the desired temperature.
Check with a thermometer to ensure that the water reaches 55 degrees Celsius. Incubate the sample in the water bath, and preheat the pipette tip on the edge of the hot plate for two minutes or longer before the blotting experiment. Glow discharge a holey carbon supported grid at 25 milliampere for 30 seconds.
Place the vitrification filter paper into the vitrification apparatus chamber no sooner than five minutes before the blotting experiment. Incubate the tweezers with the grid in the vitrification apparatus for two minutes or longer. Build the ethane container with ethane according to standard procedures, ensuring that the ethane does not overflow.
Use a pipette to apply seven to nine microliters of the sample to the grid. Then wait for one to two seconds, blot for one to one and a half seconds, and quickly plunge the sample into liquid ethane. Transfer the grid from liquid ethane to the cryo box stored in liquid nitrogen.
Clip the grids and unload them to acquire electron microscope or Cryo EM instrument. Use the display screen of the Cryo EM instrument and the low dose function of the software, to screen the ice condition on the grid and the distribution of the sample on the grid. If the quality of the resulting grid is not good, repeat the process of grid preparation with varied conditions such as waiting time, blotting time, et cetera.
If the quality of the resulting grid is good, repeat the same steps to make grids at a different temperature. Transfer the good quality grids to a high resolution Cryo EM instrument. Perform data collection and data analyses according to established procedures.
In the case of the successful grid, an ice gradient formed with thicker ice at the top left of the grid and thinner ice at the bottom right. Blue and green boxes suitable for data collection were observed in the successful grid. A bright contrast was observed in the other grid.
Indicating a thin layer, of ice or an absence of ice. Only two squares were found to be suitable for data collection in the second grid. One of the grids consisted of ice mostly in the crystalin form, which was unsuitable for data collection.
On the other hand, the other grid showed that the ice layer was mostly in an amorphous state suitable for data collection. The preparation of the as a container and others need to be complete according to a time point. Time control and and finishing the experiment quickly and accurately are most important.
Depending on the results obtained using this protocol, the researcher may have to be more careful in setting up the simple conditions.
