Computational protocol: Structure of the Drosophila nucleosome core particle highlights evolutionary constraints on the H2A-H2B histone dimer

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Protocol publication

[…] Diffraction data were collected at ESRF beamline ID14-3 (λ = 0.931 Å) on a MAR CCD detector and processed with XDS and programs of the CCP4 suite. Crystals obtained using described conditions were isomorphous to the Xla-NCP147 crystal form (Table ). The Xla-NCP147 structure (pdb id 1KX5) minus the N-terminal histone tail residues was used as a starting model. Positioning this model into the Dm-NCP147 unit cell resulted in a crystallographic R-factor of 0.40, which dropped to 0.32 upon rigid body refinement using CNS. A further round of restrained coordinate and B-factor refinement reduced this to 0.276 (Rfree = 0.301). Differences between the Drosophila and Xenopus structures were readily apparent in a 2Fo − Fc map calculated using phase information from the Xla-NCP147 atomic coordinates. Iterative rounds of manual model building using O and CNS refinement were carried out to incorporate amino acid substitutions, ions, and water molecules, and to rebuild the histone tails. The structure was refined at 2.45 Å to a final crystallographic R-factor of 0.229 (Rfree = 0.262) and good geometry. […]

Pipeline specifications

Software tools XDS, CCP4, CNS
Applications Small-angle scattering, Protein structure analysis
Organisms Xenopus laevis, Drosophila melanogaster, Homo sapiens
Chemicals Amino Acids