The overall goal of this procedure is to identify the most optimal 2D crystallization conditions for the structured determination of membrane proteins by electron crystallography. This is accomplished by first using a compatible detergent during purification to solubilize the protein from the native lipid bilayer. The second step of the procedure is to add exogenous lipid and remove detergent via dialysis of the protein lipid detergent mixture, resulting in 2D crystals under carefully controlled conditions.
The third step of the procedure is to flash, freeze the samples into a vitreous state. The final step of the procedure is to collect data by cryo em. Ultimately, results can be obtained that yield the final structure of the protein through computational image processing of the data.
Hi, I am from the laboratory. I burry e School of Biology at the Georgia Institute of Technology. Hi, I am Tina dreaded.
I'm from the Berry Lab in the school of Chemistry and Biochemistry and the Schmidt Kray Lab in the school of biology at the Georgia Institute of Technology. And I'm Matt Johnson from the laboratory of Ingleborg Schmidt Cry in the school of biology at Georgia Institute of Technology. Today we will show you a procedure for assessing 2D choral ation trials by em.
We use this protocol in our laboratory to identify and optimize 2D crystallization conditions. So Let's get started To begin this procedure, negatively stained samples on carbon coated 400 mesh copper transmission electron microscopy or TEM grids are prepared. Pipette two microliters of the protea liposome sample under a carbon covered TEM grid and incubate for 60 seconds.
If the sample does not distribute evenly, gently swipe it with the side of the pipette tip. Next block from the edge with a torn piece of watman Number four filter paper. This ensures optimal removal of liquid without excessive removal of protea liposomes, and helps to preserve the delicate carbon film.
Immediately following blotting. Apply two microliters of the 1%urinal acetate stain after 30 seconds, blot from the edge of the grid. Again, if the sample contains high concentrations of viscous glycerol or sucrose, typically in the range of 10 to 20%several cycles of washing of the grid with a glycerol or sucrose free buffer before negative staining can be helpful to allow for proper staining with the urinal tate solution.
Once the grids have been prepared, electron microscopy is used to visualize the sample at low magnification to assess for membrane occurrence distribution and morphology and overall grid quality. Then high magnification is used to obtain images and perform RIA transforms of the images to identify 2D crystals. To begin low grid in the electron microscope sample holder, to get a first impression of the average membrane distribution, use an intermediate magnification of 2000 to 10, 000 x.
Note the extent of distribution of membranes, their morphology and size in a laboratory notebook. Record representative views with a CCD camera. Next, if necessary, observe the samples with low magnification in the range of roughly 400 to 800 x to get an overview of the grids.
This can provide valuable information to evaluate the grid preparation by revealing problems with sample concentration, uneven protea, liposome distribution, and partial breakage of carbon film. Identify a grid area of interest at either low or intermediate magnification. Then use high magnification to evaluate possible order in different membranes.
This is critical at the early stages of 2D crystallization trials. In order to identify promising protea liposomes with well-ordered areas and to verify reproducibility during later experiments to determine the optimal high magnification setting for screening 2D crystals start with a magnification between 50, 000 and 60, 000 x. Depending on the 2D crystal dimensions and unit cell size, the high magnification setting may be decreased to as low as 30, 000 or raised as high as 80, 000.
Here on an adjacent location To the image area of interest, we defocus by approximately minus 400 nanometers or higher if there is uncertainty as to whether the area of interest is indeed a membrane. Inspect the sample for pieces of carbon film MICA or other artifacts that could be mistaken for protea liposomes. Also observe the edges to discern typical membrane folding and morphology.
Next, inspect the area of interest with a CCD camera. By collecting a CCD image at the optimal high magnification setting, the lattice of a smaller and or mostly hydrophobic membrane protein may not be observable by visual assessment of the CCD image itself. In this case, the entire image is used for an online foer transform or ft.
However, if the ordered array is small, this FT will contain a significant amount of noise to improve the signal to noise ratio, reduce the boxed image size, move the reduced box over the image, and perform a live ft, adjust the gamma function of the live FT for optimal identification of ordered arrays. An overly high gamma value can obscure spots due to noise contributions while an excessively low value will prevent weaker spots from being identified. Anticipate the resolution of the negatively stained 2D crystals to be roughly 15 angstroms at a DFO of minus 400 nanometers.
Expect to identify one to three orders of spots. Note the sharpness of the spots and any mosaic. 2D crystals of a small 18 kilodalton membrane protein are up to several microns in size.
Spots on the FTS sharp and easily identified movement of the live FT box shows that the lattice is continuous without mosaic. The lattice of a larger protein with a more extensive soluble domain can be identified on the small screen of the T-E-M-C-C-D image collection and FT are necessary to get a better assessment of the lattice quality, including characteristics such as mosaic noise in the FT of an unordered protea. Liposome may be mistaken for weak spots to determine whether the spots are due to small protein 2D crystals or noise.
Move the box for the live ft. If the spots disappear immediately they are noise, but if left unchanged over even a small area, crystals are likely present since lipid crystals display a distinct morphology and ft. These lipid crystals can also be recognized by visual inspection of an image and consistent unit cell sizes.
Precipitation can occur in initial trials. Inspection at high magnification is used to distinguish between protein precipitation and lipid aggregates. In the case of lipid aggregates, reconstitution might might actually have occurred and the next experiments will only require adjustment of parameters necessary to increase membrane size and produce order rather than to induce reconstitution at 30, 000 to 50, 000 x.
The edges of these dark structures reveal them to be composed of membranes with no protein. Precipitation samples under optimal conditions will contain a large percentage of 2D crystals. It is not necessary to aim for a homogeneous appearance of membranes as the largest and most well-ordered 2D crystals are selected visually for data collection.
These types of samples will be easily recognized when the crystallization such samples are used for cryo EM data collection to result in the maximum number of high resolution images. We will just show you how to screen for 2D crystal of small memory proteins by TEM when doing this procedure. It's important to remember to be thorough to not overlook a promising crystallization condition as you've already invested a significant amount of time and effort up to this point.
So that's it. Thanks for watching and good luck with your experiments.
