I am trying to collect the cryoEM data of a protein. The protein is ~25 kDa but oligomerizes with 10 or 12 molecules forming a ring of homodecamer or homododecamer. In the negative staining with manual blotting, the ring structure is preserved. However, when I prepare the grid for cryoEM using a vitrobot, the ring structure is seen to be dissociated while screening the grid. I have tried different concentrations of the protein as well as different setting for vitrobot but I could not obtain the ring structure of the protein complex as seen in the negative staining. How can I get the similar ring structure in cryoEM grid as seen in the negative staining grid?
Hey,
Does your protein stays in oligomeric form until before deposing on the grid? ( at the concentration needed for your grids?) If it is the case, I guess your complex does not withstand the harsh conditions of blotting and freezing. In this case, you can continue trying different blotting conditions, incubation times, a blot-less method of grid preparation. Another option would be to stabilize your complex : check these papers here :
https://pubmed.ncbi.nlm.nih.gov/20887855/
Article GraDeR: Membrane Protein Complex Preparation for Single-Part...
I wonder if your sample is somewhat unstable and as mentioned, can't withstand freezing and blotting. Have you tried using a different buffer? You may be able to find some cosolutes that are more stabilizing to your desired structure.
Thank you Jorgaq and Meranda. I will try to optimize buffers and blotting conditions. I hope your suggestions will help.
I would reduce blot force, and increase blot time to compensate (maintain ICE gradient). Also try different grid supports, we use lacey carbon with ultra thin carbon if we are having problems with sample in ICE (quantifoil holes), can be a good indicator of what's going on.
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