Cryo-EM analysis of the Kap114/Nap1 ₂ /H2A-H2B ternary complexes. (A) Top left: Schematic of how the cryoEM sample was assembled with its SEC chromatogram below, with fractions 1–6 colored red to purple. The typical elution volume of an uncrosslinked complex of Kap114, Nap1₂, and H2A-H2B is ∼11 ml (dotted line). Each fraction was analyzed by mass photometry, and the data are plotted as in Fig. S2 D. Protein aggregates beyond 600 kDa were not displayed. Proteins in the ∼340–380 kDa peak may be crosslinked complexes of K/N₂/H with K/H; such a large complex was not observed in AUC or SEC-MALS studies. Fraction 6, most enriched with the 1:1:1 Kap114:Nap1₂:H2A-H2B complex and has the least large aggregates, was used for cryo-EM grid preparation. (B) Distribution of the ∼1 million particles produced by the cryo-EM data: 38.7% are Nap1₂, 36.0% Kap114•H2A-H2B, and 25.3% ternary complex of Kap114/Nap1₂/H2A-H2B (see more statistics in Table S1). The small population of ternary complex is likely due to destabilization of the assembly by excess H2A-H2B as shown in Fig. S1. Ternary complex particles were classified into two evenly divided classes that produced two high-resolution maps and structures. Left to right: Final consensus maps for Nap1₂ (EMD-44095; cyan) overlayed with the final model (9B23; cyan), consensus (EMD-44122; gray) and local refined (EMD-44121; cyan) maps for Nap1₂•Kap114•H2A-H2B overlayed with the final model (9B31; cyan•gray•yellow-red), and consensus (EMD-44140; gray) and local refined (EMD-44137; cyan) maps for Nap1₂•H2A-H2B•Kap114 overlayed with the final model (9B3F; cyan•yellow-red•gray). Consensus maps are also shown below colored by local resolution. Nap1₂•Kap114•H2A-H2B resembles the Kap114•H2A-H2B structure (8F0X), with Nap1₂ contacting Kap114 h19loop via the β-hairpin. This structure may represent a ternary complex that is falling apart or it may be one configuration of a dynamic ternary complex ensemble. (C) Left to right: Phenix map-to-model FSC curves for the consensus map for Nap1₂, or the composite maps for Nap1₂•Kap114•H2A-H2B (EMD-44120) and Nap1₂•H2A-H2B•Kap114 (EMD-44136). (D) 3D angular distribution of the particles that were used for reconstructions of the consensus maps above. The left orientation is same as in B. (E) Directional FSCs unmasked and masked by cryoSPARC refine mask of the consensus maps above. (F) The Nap1₂•H2A-H2B•Kap114 structure overlayed onto the local refined map (gray mesh), zoomed into the Kap114-H2A-H2B, Kap114-Nap1₂ and Nap1₂-H2A-H2B interfaces.