Computational protocol: Structural polymorphism in the L1 loop regions of human H2A.Z.1 and H2A.Z.2

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Protocol publication

[…] The crystals of human nucleosomes containing H2A.Z.1 and H2A.Z.2 were grown by the hanging-drop method at 293 K. The hanging drop was formed by adding 1 µl of each nucleosome (at a concentration of 2.5–4.0 mg ml−1) to 1 µl crystallization solution (20 mM potassium cacodylate pH 6.0, 50–70 mM KCl, 75–105 mM MnCl2). For data collection, the crystals were harvested in reservoir solution containing 30% 2-methyl-2,4-pentanediol and 2% trehalose and were flash-cooled in a stream of nitrogen gas at 100 K. Data sets were collected on the BL41XU and BL38B1 beamlines at SPring-8, Harima, Japan. The data sets were processed and scaled using the HKL-2000 program suite (Otwinowski & Minor, 1997). All nucleosome crystals belonged to the orthorhombic space group P212121 and contained one nucleosome per asymmetric unit. Unit-cell parameters are provided in Table 1.The structures of the H2A.Z.1 and H2A.Z.2 nucleosomes were solved to 3.07 and 3.20 Å resolution, respectively. The data were processed using the CCP4 suite of programs (Winn et al., 2011). The structures of the nucleosomes were determined by molecular replacement with Phaser (McCoy et al., 2007) using the coordinates of the human nucleosome structure (PDB entry 3afa; Tachiwana et al., 2010) as the search model. To eliminate the model bias from the electron-density map, we removed the Lys36–Arg42 region of H2A from the model prior to the initial round of refinement. All refinements were performed using PHENIX (Adams et al., 2010). After rigid-body refinement, the model was refined by iterative rounds of xyz-coordinate, real-space, individual B-­factor and occupancy refinements and manual model building using Coot (Emsley & Cowtan, 2004). For all refinements, secondary-structure restraints and noncrystallographic symmetry (NCS) restraints were applied between chains A and E, chains B and F, chains C and G and chains D and H. The Ramachandran plots of the final structures showed no outlying residues, as assessed with the MolProbity program (Chen et al., 2010). A summary of the data-collection and refinement statistics is provided in Table 1. All structure figures were created using PyMOL (Schrödinger; http://www.pymol.org). The atomic coordinates of all nucleosomes have been deposited in the RCSB Protein Data Bank, with codes 3wa9 and 3waa for the H2A.Z.1 and H2A.Z.2 nucleosomes, respectively. […]

Pipeline specifications

Software tools HKL-2000, CCP4, PHENIX, Coot, MolProbity, PyMOL
Application Protein structure analysis
Organisms Homo sapiens