The overall goal of this procedure is to determine the structure and organization of mitochondrial proteins in C two. This is accomplished by first generating a frozen hydrated electron microscope grid containing the sample. Next, a dose limited tilt series of the sample is recorded in an electron cryo microscope.
Then the tilt series is processed to generate a tomographic volume. Finally, the protein densities are averaged to determine the structure of the proteins and the membranes are segmented to reveal their 3D structure. By repositioning the averages of proteins back into the Tom Grahams, the structure and organization of proteins in the membrane is revealed.
So we do electron cry tomography of mitochondria because we want to understand how they work at the molecular level. The main advantage of this technique over other methods, such as electron microscopy of thin plastic sections is that the resolution is higher. The material is not chemically fixed in any way, and the molecular detail of the proteins are preserved.
The technique of electron crytography can take some time to master, but experienced users can acquire five to six good quality TOM grounds per session. The most difficult part of the procedure is to produce frozen hydrated grids of optimal ice thickness After isolating and pelleting mitochondria, according to the text protocol, use 250 millimolar T trioses in 10 millimolar HEPA buffer to re suspend the pellet to a concentration of approximately five milligrams per milliliter of total protein glow discharge. Wholly carbon EM grids carbon side up in a vacuum device according to manufacturer's instructions, liquefy a few milliliters of ethane by directing a stream of ethane gas onto the inner side of a liquid nitrogen cooled aluminum container.
Use a pair of tweezers to pick up a glow discharge DM grid and place it on the bench mix protein, a conjugated gold fiducial suspension, one-to-one with the mitochondrial suspension, and immediately apply three microliters of the solution to the grid. Next, place the tweezers in a vitrification device such as a homemade guillotine with a wedge of filter paper blot off the excess liquid from the grid and release the trigger to immediately plunge the grid into liquid ethane. Then to transfer the grid from liquid ethane into liquid nitrogen.
Remove the excess ethane from the grid by placing the tip of a fresh filter paper wedge into the liquid ethane. As the liquid rises, gently drag the grid up the filter paper, keeping it below the liquid front, and then immediately transfer it into liquid nitrogen to store for later use. Place the grid in a grid box and store in a liquid nitrogen filled doer to record a tomographic tilt series.
Begin by ensuring that the liquid nitrogen doers are full, and check that the temperature of the specimen stage and grid transfer device are below 100 kelvin. Confirm the electron microscope is well aligned by acquiring an image of a test grid and checking the quality of the thorn rings T tho rings should be round and extend to the edge of the image. Next, insert a grid with frozen hydrated mitochondria.
Then in search mode, search for areas of appropriate ice thickness and specimen quality. Take a six second search image of promising areas to determine suitability for data collection. After locating a good specimen area, tilt the stage plus or minus 60 degrees to determine the maximum tilt range that is available without obstruction of the exposure or focus area by grid bars or ice crystals on a nearby ice filled hole of similar appearance, change to exposure mode and adjust the beam intensity or image acquisition time.
So each recorded image has an electron dose of 30 to 50 electrons per pixel for CCDs or six to eight electrons per pixel per second. For direct electron detectors, calculate the dose distribution ratio or I zero over I 60 by dividing the average electron Count for a one second image acquired at zero degrees by that of a 60 degrees image over an empty hole. Acquire a one second image in exposure mode and note the electron count per angstrom squared.
Taking into account the dose distribution ratio, calculate the total number of images and tilt interval that can be recorded for a specific total electron dose using appropriate automatic data collection software. Set up and record a Tom Agram with the parameters just determined typical tomos. For these samples, start at plus or minus 20 degrees and go through zero degrees before reaching high tilts.
To create and segment tomos, convert the tilt series to a file format appropriate for the reconstruction software. Using a Tom Graham reconstruction program like Im mod, align the images by marking the position of the gold fiducial markers. Once aligned, generate a Tommo Graham.
Enhance the contrast of the tommo Graham by using an image filter such as the non-linear an isotropic diffusion filter distributed with IM o for visualization. Use commercially available programs to manually segment the Tom. Assign voxels corresponding to the inner and outer membrane to separate layers once assigned, create a surface to visualize the membranes.
Using the clicker option in the EM package plugin for emira, mark the location of a TP syntase particles using the marked particles as input and an appropriate software package such as the particle estimation. For electron tomography program, calculate a agram average for a resolution estimate. Calculate the foyer shell correlation between two independently determined sub tommo averages using the free visualization program.
Chimera fit known x-ray structures in into the sub Tom Agram average first manually, then automatically with the command fit as seen in this figure, manual segmentation of the membranes in a mitochondrial electron cry reveals the structure of the Christi in a mitochondria. By imaging mitochondria from yeast knockout strains that lack certain proteins. The effect of these proteins on Christi morphology can be assessed.
Shown here is a mitochondria from a yeast strain lacking a TP synthesis subunit E, which is required for the formation of a TP synthe dimers. Unlike the normal Lala Christi of wild type mitochondria, these organelles instead contain a number of inner membrane structures that are either devoid of Christi or contain small balloon shaped membrane invaginations. In Ingrams with good contrast, A TP Synthes dimers are easily visible as indicated here by the yellow arrowheads.
By segmenting the membranes, the location of the A TP synthesis now represented by the yellow sphere can be visualized in relation to the morphology of Christi. In this case, the A TP synthesis form rows of dimers along the highly curved edges of the Lo Meer Christi Sub tommo averaging allows the structures of proteins to be determined at resolutions of four nanometers or better. By fitting known x-ray structures into the sub tomo Graham average atomic models of protein complexes in their native environment can be assembled as seen.
In this example, average volumes may be placed back into the Tomo Graham in order to assist the organization of individual complexes relative to each other and to other protein complexes in the membrane. Our protocol demonstrates how you can use electron cryo tomography to determine the structure and organization of protein complexes within the inner mitochondrial membrane. This protocol can also be used to determine the structure and organization other membrane bound proteins within different cellular compartments.
Sample preparation is the key for acquiring good thermograms. The frozen hydrated grid must contain perfectly vitreous eyes of 70 to 150 nanometer thickness. Normally we screen five to six grids until you find an area of optimum ice thickness and specimen quality.
For gram collection, a 300 kilovolt transmission electron microscope with dedicated cryo stage and energy filter and camera with direct electron detector is best, but a 200 kilovolt instrument with side entry cryo stage and A CCD camera could also be used. After watching this video, you should have a good understanding of how to prepare a cry m grid. Collect a tilt series, reconstruct thermogram, and calculate a sub tommo average.
These are the basic skills required to investigate how the organization of proteins in a cell affect that cell's ability to perform biological functions such as efficient A TP synthesis. Don't forget working with liquid nitrogen and liquid Ethan can be very hazardous and safety glasses and gloves should be worn at all times.
