Computational protocol: Structure of the yeast histone H3-ASF1 interaction: implications for chaperone mechanism, species-specific interactions, and epigenetics

Similar protocols

Protocol publication

[…] Concentrated protein (10 mg/ml) was dialyzed against 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, and 1 mM DTT. Crystals were grown by hanging-drop vapor diffusion by mixing the protein 1:1 with well solution (85 mM Tris-HCl (pH 8.5), 120 mM Li2SO4, 25% PEG 4000, and 15% glycerol) at 18°C; reservoir solutions were diluted two-fold with dialysis buffer prior to sealing crystallization chambers. Crystals were harvested and flash-frozen in liquid nitrogen directly from the drop.Data were collected using the Advanced Light Source Beamline 8.3.1 at Lawrence Berkeley National Laboratory []. Diffraction data were processed using an automated MOSFLM [] procedure implemented in ELVES []. Phases were solved by molecular replacement using Phaser [] and histone-free yeast Asf1 N-terminal domain as a search model (PDB ID 1ROC) []. Model building was carried out using O [], and refinement with REFMAC, ARP [] and CNS []. The final model, which contains amino acids 2–155 of ScAsf1 and 121–131 of histone ScH3, has an Rwork of 19.6% and an Rfree of 23.9%. No density was observed for the fusion linker past the first two alanine residues and the linker was not included in final model. No residues lie in disallowed regions of Ramachandran space. The coordinates and structure factors have been deposited in the Protein Data Bank (PDB ID 2IDC). […]

Pipeline specifications

Software tools iMosflm, CNS
Applications Small-angle scattering, Protein structure analysis
Organisms Saccharomyces cerevisiae