Supplementary Figure 4: Data collection and image processing procedures for the PRC2–DiNcl35 complex
From: Cryo-EM structures of PRC2 simultaneously engaged with two functionally distinct nucleosomes
(a) For the first dataset, a subset of micrographs (top left: example micrograph showing PRC2-dinucleosome complexes in vitreous ice; green circles represent example particle picks) was used for manual picking and reference-free 2D classification (top right: example classes) to generate templates for automated particle picking using Gautomatch (Kay Zhang, MRC LMB, Cambridge, UK). Initial particle picks were reviewed and edited manually to exclude ice and aggregates and increase the number of good picks. A final set of 104,836 particles were subjected to 3D classification using an initial model that was manually created based on the 2D class averages obtained after manual picking. (b) Particle images from automated picking were used for 3D classification of the second dataset, bad picks were sorted out by two rounds of 3D classification, resulting in a subset of 38,400 particles from a total of ~2,000 micrographs after sorting out bad micrographs. (c) The smaller, third dataset was processed analogously to the second dataset.
