Structural Determination of HIV-1 IN CCD Mutant

The HIV-1 IN CCD (residues 50–212) containing the F185H mutation was expressed and purified as described (Sharma et al., 2014 (link)). The protein was concentrated to 8 mg/ml and crystallized using hanging-drop vapor diffusion method with a crystallization buffer consisting of 100 mM sodium cacodylate pH 6.5, 100 mM ammonium sulfate, 10% (w/v) PEG 8000, and 5 mM DTT. Crystallization drops were prepared using an equal volume of protein and well solution. Crystallization trays were prepared on ice at room temperature and then transferred to 4°C for storage. Crystals formed within one week and continued to grow thereafter in size. Crystal data were collected on a Rigaku Micromax-007 at 100 K. Data were integrated and scaled using HKL3000 (Minor et al., 2006 (link)) and Scalepack (Otwinowski and Minor, 1997 (link)). Phaser (McCoy et al., 2007 (link)) in the PHENIX suite (Adams et al., 2010 (link)) was used to run molecular replacement using Protein Data Bank code 4O55 as a search model (Sharma et al., 2014 (link)). Phenix.refine (Afonine et al., 2012 (link)) was used for data refinement, and manual refinement was done in Coot (Emsley et al., 2010 (link)). The coordinates are deposited in the Protein Data Bank under accession codes 6NUJ. The data and refinement statistics are given in Supplementary file 1 (Table S1).