Computational protocol: Structure of HsaD, a steroid-degrading hydrolase, from Mycobacterium tuberculosis

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[…] Purified recombinant HsaD protein (in 100 mM sodium phosphate pH 7.4) was concentrated to 10 mg ml−1 with an Amicon ultracentrifugation concentrator (Millipore, Watford, Hertfordshire). The crystals described in this paper were grown at 292 K by the sitting-drop vapour-diffusion method. For crystallization, 150 nl concentrated HsaD solution was mixed with 150 nl precipitant [30%(w/v) PEG 3000, 0.1 M CHES pH 9.5] and the volume of precipitant in the reservoir was 100 µl. Crystals typically grew within 3–5 d.Crystals were briefly transferred to a cryoprotectant solution [a 3:1(v:v) mixture of precipitant and glycerol] prior to flash-freezing in liquid nitrogen. Diffraction data were collected from one plate-shaped crystal of approximate dimensions 50 × 50 × 10 µm at beamline IO4 at the Diamond Light Source, Oxfordshire, England. Data from 178 images (oscillation range 0.5°) were indexed and integrated with MOSFLM (Leslie, 1992) and scaled with SCALA (Evans, 2006). Initial phases were determined by molecular replacement with the program Phaser (Read, 2001) by using an ensemble of the MCP hydrolases from Burkholderia xenovorans LB400 (PDB code 2og1; Horsman et al., 2006) and Rhodococcus jostii RHA1 (PDB code 1c4x; Nandhagopal et al., 1997) as search templates, with non­conserved residues truncated to the Cβ atom with the program CHAINSAW (Schwarzenbacher et al., 2004). Initial model building was performed with ARP/wARP (Morris et al., 2003) and was followed by iterative cycles of manual building with Coot (Emsley & Cowtan, 2004), refinement with REFMAC (Murshudov et al., 1997) or phenix.refine (Adams et al., 2002) and model-quality checking with MOLPROBITY (Davis et al., 2007). The protein model was solvated and the waters checked with phenix.refine and Coot. The stereochemical quality of the final model was assessed with the programs MOLPROBITY and PROCHECK (Laskowski et al., 1993). Data-collection and refinement statistics are shown in Table 1. […]

Pipeline specifications

Software tools ARP/wARP, MolProbity, PROCHECK
Application Protein structure analysis
Organisms Mycobacterium tuberculosis, Dipturus trachyderma
Diseases Tuberculosis
Chemicals Carbon, Cholesterol, Steroids