I’m a structural biologist by using Cryo-EM.
My protein always form a stable complex (GPCR-antibody). But on cryo-em grid, my particles seem to be dissociated.
I use a 200kV microscope comprising of K3 detector. And, I usually collect micrographs at 60 total dose.
I have a question. My total dose usually high or low?
What is the relationship between total dose and particle dissociation?
If reducing the total dose, expect to strongly form the complex ?
HI Shinjw,
I think you can try to add crosslinker, such as glutaraldehyde, this will help to stabilize your complex.
Good luck!
dose has no effect on particle dissociation. 60 total is fine, only a bit high. it is a process that occurs prior to or during the grid vitrification. most likely caused by Air-water interface AWI. you can look this up and find lots of information about causes and potential circumventions - including addition of detergents, changes to blotting procedure, protein concentration, and yes purification in the presence of crosslinkers like glutaraldehyde.
Keep in mind that if you’re looking at a dose-fractionated image that has been aligned to produce a final single frame, your frame is likely dominated by the lower-dose images, because they incorporate most of the high resolution data, and to my knowledge, most frame-alignment software will dose-weight the images to preserve the high-resolution information.
If you are curious as to the exact consequence of dose, you can simply align the first X frames to see what your particles look like at that dose.
60 seems fine to me.
I doubt it. But again, it should be simple enough to check. You should be able to see the particles at 30. At least well enough to do some 2D averaging.
One question is - did you alter the buffer for vitrification? i.e. lower salt concentration, or glycerol concentration, etc?