Hi,
I am also experiencing problems with a membrane protein with a tight fit in a nanodisc. It seems that after ab initio reconstruction I can see the an obvious scaffold protein with central density but as soon as I try any refinement of the particles I get very little definition of any disc or central density. The refinements result mostly in noise. I have tried to make a mask around the central density to exclude the scaffold seen in the ab initio reconstruction but it makes no difference.
Is this a problem exclusively with my particle quality which I suspect is not good or am I doing something wrong? I think there is a fair amount of movement of the nanodisc relative to the membrane protein which I think it making this a very tricky sample. In addition I currently have some orientation bias in my sample. I am preparing more grids also but if anyone has anymore tips for how to work with the data I have and any future data that would be much appreciated.