Fig. 1. CENP-N mediates CENP-A nucleosome stacking in vitro.
a, A model of two CENP-A nucleosomes (α satellite DNA) connected by two copies of CENP-N was fit into the density map. Protein identity is indicated by color codes. Arrows highlight the weak density attributed to a second CENP-N on the other side of the CENP-A nucleosomes. b, Simulation plots of nucleosome stacks in the absence or presence of CENP-N. This plot used two coordinate points from each nucleosome, namely the geometric center of the C1′ atoms from nucleotides located at the dyad and opposite of the dyad (Extended Data Fig. 4 for illustration). The C1’ atoms of every nucleotide in one nucleosome, pictured as the bottom nucleosome of each graph in Fig. 1b, were used to align each frame of the trajectory. The dyad and opposing points in the other nucleosome, pictured as the top nucleosome of each graph in Fig. 1b, were plotted to depict the relative sampling of each stacked-nucleosome system. c, SV-AUC (enhanced van Holde–Weischet plots) for CENP-A (CA) or H3 mononucleosomes (MN) in complex with CENP-N1–289 (CN) or full-length CENP-N/CENP-L (CN + CL). d, FRET analysis of CENP-A mononucleosome (CA MN) interactions in the absence or presence of CENP-N. The donor is CENP-A mononucleosomes containing Alexa 488-labeled H2B; the acceptor is a CENP-A mononucleosome containing Atto N 647-labeled H2B (250 nM donor and acceptor nucleosome concentrations were used). FRET intensity changes in dependence of [CENP-N]. The final NaCl concentration was 70 mM. Error bars are from four independent measurements of two biological replicates. Data are presented as mean values ± s.d.