Supplementary Figure 1: Experimental strategy and reconstitution of the PRC2–DiNcl complexes
From: Cryo-EM structures of PRC2 simultaneously engaged with two functionally distinct nucleosomes
(a) Schematic representation of the generation of substrate dinucleosomes. See methods section for details. ΦK27me3 = pseudo trimethylated lysine. (b) Dinucleosome ligation and purification. A native PAGE gel shows mononucleosomes before ligation (Nclmod and Nclsub), after ligation (Lig), and following preparative native PAGE purification (DiNcl). (c) Left, SDS-PAGE of the purified PRC2 complex used in this study. 4-20% gradient gel, stained with coomassie. Right, electrophoretic mobility shift assay of dinucleosome binding to PRC2 under the conditions used for cryo sample preparation. 1.2 µM dinucleosomes with 35 bp (DiNcl35), 40 bp (DiNcl40) or 30 bp (DiNcl30) linker lengths were incubated with 1.6 µM PRC2. Gels in (b) and (c) were stained with SYBR gold. (d) Reference-free 2D classes of negatively-stained EM samples of PRC2-DiNcl35 (left, after an initial round of 2D class averaging to include only classes with two nucleosomes) or PRC2 alone as a control (right).
