I know a small protein (40 kD) can bind to ribosome and affects ribosome translation process (We demonstrated it by biochemical experiment). So, I want to solve the structural mechanism of how this protein affects translation process.
I put this small protein with ribosome and collect 6000 micrographs by CRYO ARM 300, in the further refine, I have never found a extra density in the ribosome that maybe this small protein.
So anyone have a advice to help me to solve this problem. Thanks in advance!
It might not bind tightly. This will mean that in your prep you might have only partial occupancy, which means a lot of your ribosomes will be “normal”. That is a classification game, which you will need to do unmasked or you risk masking it out.
What ratio did you mix the ribosome and inhibitor protein? You will probably need excess of the small protein relative to the ribosome.
Where do you expect it to bind?
Is it binding to the ribosome itself or the RNA and affecting translation that way?
How long did you incubate after mixing, before vitrification?
What evidence do you have for tight binding, rather than just transient interaction?
Is it a stable complex? Can you run on a size exclusion column and see a change in the elution profile compared to just ribosome?
Do you have evidence from maybe another technique such as mass photometry?