Computational protocol: Structural and mechanistic insights into regulation of HBO1 histone acetyltransferase activity by BRPF2

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Protocol publication

[…] Prior to crystallization, the HBO1–BRPF2 complex was mixed with AcCoA (Sigma) at a molar ratio of 1:3. Crystallization was performed using the sitting drop vapor diffusion method at 16°C by mixing 1 μl protein solution (10 mg/ml) with 1 μl reservoir solution containing 2% Tacsimate (pH 8.0), 0.1 M Tris–HCl (pH 8.5) and 12% (w/v) polyethylene glycol 3350. The crystals were cryoprotected with the reservoir solution supplemented with 20% (v/v) glycerol and then flash frozen in liquid nitrogen. The diffraction data were collected at −175°C at beamline 19U1 of National Facility for Protein Science in Shanghai, China and were processed, integrated and scaled together with HKL3000 (). The structure of the HBO1–BRPF2 complex was solved using the molecular replacement (MR) method as implemented in Phenix () using the structure of the MYST domain of human MOF (PDB code: 3QAH) () as the search model. Residues 39–62 of BRPF2 were clearly defined in the initial MR-phased electron density map and the model was manually built with COOT (). Structure refinement was performed using Phenix () and Refmac5 (). Structural analyzes were carried out using programs in CCP4 (). The structure figures were generated using PYMOL (http://www.pymol.org). Statistics of the diffraction data, structure refinement and quality of the structure model are summarized in Table . […]

Pipeline specifications

Software tools PHENIX, Coot, REFMAC5, CCP4, PyMOL
Application Protein structure analysis