Figure 2. CryoET of cryoFIB-SEM lamellae containing yeast cells with intracellular protein aggregates recruiting HSP104-GFP reveals that the unfolded proteins are compacted into globular densities.

(A). Z-directional, maximum intensity projection of CACM z-stack images of yeast cells expressing Hsp104-GFP. (B) The last cryoFIB-SEM MAV slice, right before creating the lamella. (C) Superposition between (A) and (B) to correlate fluorescent signals with areas seen by cryoFIB-SEM to select optimal regions on the lamella that both contain the target and are amenable for subsequent cryoET experiments. (D) Top and (E) slanted views of the final milled lamella (white arrow highlights an estimated thickness of ~260–300 nm). (F) Slice (~3 nm thick) through a representative cryoET tomogram collected from a region with a bright fluorescent punctum and corresponding annotation of features (bottom row) in different colors (green:inclusions containing Hsp104-GFP; yellow:a multivesicular body made of lipid membranes; pink:ribosomes. Scale bars: A-C, 5 μm; D,E 2.5 μm; F, 100 nm).