(A) Interaction of JARID2 K116me3 (magenta) with EED (light blue) in the two conformational states sampled by PRC2-AJ106. JARID2 K116me3 sits in the middle of an aromatic cage (F97, Y148, and Y365) with hydrogen-bond interactions (R414:F117 and W364:R115) that stabilize the JARID2 peptide backbone. In the compact active state, additional interaction between EZH2 D136 and the backbone amide of JARID2 K116 helps position the EZH2 SRM helix (gold) next to the EZH2 SETdomain. (B) The EZH2 SETdomain of PRC2-AJ106 is in a similar conformation in both states (compact active, orange; extended active, blue; Cα root mean square deviation = 0.74 Å; ) and is bound to a substrate peptide (cyan). Inset, density of substrate observed in our cryo-EM reconstruction shown with a model for JARID2 residues 114 to 118. (C) Cryo-EM density of the PRC2-AJ119 EED and EZH2 SETregions with ribbon representations of EED (light blue) and SET (dark blue), showing the absence of both the stimulatory fragment bound to EED and the substrate bound to the SETdomain (red dashed circles). (D) AEBP2 (residues 232 to 295, red), shown in surface representation, connects RBAP48, the SUZ12 N-terminal region, and the EZH2 SETdomain. (E) Map versus model of the AEBP2 region that binds RBAP48 at the histone H3 binding site. (F) View of the RBAP48 WD40 domain showing, in stick representation, the conserved amino acids in RBAP48 (violet) interacting with K294 and R295 of AEBP2 (red). The overlay of the crystal structure of the Drosophila homolog of RBAP48 (NURF55) bound to unmodified histone H3 peptide [PDB ID, 2YBA (); green] highlights the similarity of the binding mode between AEBP2 and histone H3, with all key residues in identical conformations; only the AEBP2 (H3) residues R295 (R2) and K294 (K4) are shown for clarity. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.