Description
The total electron dose (e-/A2) is a critical parameter in a single particle cryo-EM method. A higher dose allows to achieve a higher signal to noise ratio, which should give a better quality of final macromolecule reconstruction, but also causes a bigger beam-induced motion. Too high dose can destroy the most sensitive parts of observed particles. In general, it is known how to determine the optimal dose for proteins and DNAs for obtaining a high resolution of reconstruction. Unfortunately, for RNA radiation damage problem is not clearly understood. In my PhD research, I will try to find answers to a few essential questions about the resistance of nucleotides to beam radiation:
- Which nucleotide is the most sensitive to beam radiation and which one is most resistant?
- Do RNA nucleotide/base pairs react differently to radiation damage than unpaired RNA nucleotides?
- Which pair of RNA nucleotides is the most sensitive/resistant to radiation damage?
- Are non-canonical pairs more resistant to radiation than canonical ones?
- Do bound protein partners influence the beam affected nucleotide pairs?
To reach these aims, I’ve collected a dataset on the transmission electron microscope (TFS Krios G3i) for human type Ribosome and E.coli Ribosome with dose 160 e-/A2 (160 frames) and 80 e-/A2 (80 frames) accordingly. Next, I sliced each movie for a sequence of frames. This procedure should deliver information on the impact of dose on the degree of particle destruction and how beam-induced motion changes the final reconstruction resolution.
