Structural Basis of RCC1-Nucleosome Complex

The complex of Drosophila RCC1(2–422) and nucleosome core particles containing Xenopus core histones and the 147 bp Widom 601 sequence were purified by size exclusion chromatography and crystallized against 25 mM sodium acetate pH 5.5, 25 mM sodium citrate, 1 mM DTT and 6–7% polyethylene glycol monomethyl ether 2,000 (PEG MME 2,000) at 21°C. Crystals were soaked in 25 mM sodium acetate pH 5.5, 25 mM sodium citrate, 1 mM DTT, 5% ethanol, 10% PEG MME 2,000 containing increasing concentrations of polyethylene glycol 400 (0 to 24% in 2% increments) before flash cooling in liquid nitrogen. Diffraction data were collected using an ADSC Quantum 315 CCD detector at Advanced Photon Source’s NE-CAT beamline 24-ID-E, and the data was processed using the HKL-2000 program suite³⁸ . The structure was solved by molecular replacement using Phaser software^{39 (link)} and a search model containing Drosophila RCC1, the histone octamer with tails removed, and the 147 bp human αsatellite DNA, each treated as a rigid body. Crystallographic refinement was carried out using REFMAC5^{40 (link)} and PHENIX^{41 (link)} together with manual model building in COOT^{42 (link)}. The structure was refined to 2.9 Å resolution with R_work/R_free of 17.49/21.55%. All molecular graphics were prepared using PyMOL⁴³ .