(a) A plot of motion between neighboring frames when frame exposure was set to 0.075 second (blue) and 0.15 second (red, by average two adjacent sub-frames to produce a sub-frame that is equivalent to the 0.15 second frame exposure time). (b) Red: FSC curve of an archaeal 20S proteasome 3D reconstruction determined from a dataset of ∼36,000 particles collected with a TF20 electron microscope operated at 200kV acceleration voltage. The defocus range was set between 0.6μm and 2.6μm. Blue: FSC curve of a 3D reconstruction using a subset of 3,000 particles with low defocus (0.6μm to 1μm). Purple: FSC curve of a 3D reconstruction of using another subset of 3,000 particles high defocus (2.0μm to 2.6μm). (c) Ribbon diagram of a part of archaeal 20S proteasome. (d) Same region of the 3D reconstruction determined from the entire dataset, corresponding to red FSC curve in (a). (e) Same region of the 3D reconstruction determined from the subset of 3,000 particles with only low defocused particles (blue FSC curve). (f) Same region of the 3D reconstruction determined from a subset of 3,000 particles with only high defocused particles (purple FSC curve). All maps (d, e and f) are shown at the same normalized density level, σ = 4.