Hi,
I am working on a 150 kDa protein that is almost fully embedded in the membrane. It purified nicely and I am rather sure that every micell is has a protein. I collected a million particles in rather good quality, but I am struggling to classify them. Would you have suggestions what to try? I do think I see a little bit of protein density in the 2D class averages, but I am really struggling to get a useful model from that. Does anyone have experience with proteins that are completely embedded in the membrane?
Hi David,
I dealt with a similar case recently (Sauer et al. eLife 2020). Essential the strong but low-resolution signal of the micelle was dominating the alignment. Keys to resolving this for us were: thin ice to maximize signal to noise, a fiducial marker (Fab), or making the micelle “smaller” using an amphipol.
Hey David!
I am encounting the same/ similar problem. I have a small membrane protein as a dimer or trimer in a micelle. Based on the purification I am fairly sure that the micelles contain my protein of interest. However I am struggling to get them into nice 2D classes with secondary structure features. Were you able to tackle the problem? Would be helpful for me
cheers
Sa
