Computational protocol: Crystal Structures of the Kinase Domain of the Sulfate-Activating Complex in Mycobacterium tuberculosis

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[…] Crystals of the CysC•ADP complex were obtained at 20°C using vapour diffusion. Droplets of an enzyme solution (27.5 mg/mL, pre-incubated with 5 mM ADP) were mixed in a 1:1 (v:v) ratio with crystallization buffer (0.2 M NH4NO3, 20% (w/v) PEG3350) and equilibrated against the same buffer solution. For data collection, crystals were transferred to cryoprotectant (mother liquor containing ADP and 25% (v/v) ethylene glycol) and then frozen in liquid nitrogen. Crystals of the CysC•ADP•APS complex were obtained by pre-incubating CysC at 27.5 mg/mL with 2.3 mM ADP, 1.9 mM APS and 1.8 mM MgCl2. The enzyme solution was mixed in a 2:1 (v:v) ratio with buffer containing 0.1 mM citric acid, pH 5.5, 20% (w/v) PEG 4000 and 10% (v/v) 2-propanol and the droplets were equilibrated against 500 μL of the same buffer. Crystals of the CysC•AMP-PNP•APS complex were obtained under the same conditions, except that ADP was replaced by 2.3 mM AMP-PNP. Crystals of the Cys556Ser mutant were obtained at 20°C by mixing 1 μL protein solution (40 mg/mL, 5 mM MgCl2 and 5 mM ADP) and 1 μL of the well solution (200 mM NH4NO3, 21% PEG3350). Crystals were cryoprotected by dipping them in a solution of 200 mM NH4NO3, 21% PEG3350, 5 mM ADP, 5 mM MgCl2, 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 22.5% ethyleneglycol) for 5–10 seconds and subsequent flash-freezing in liquid nitrogen.Diffraction data were collected at beamlines ID14–1 and BM14 of the European Synchroton Radiation Facility (ESRF, Grenoble, France) or beam line BL14–1 of BESSY (Berlin, Germany) from crystals in a nitrogen gas stream at 100 K, processed with iMOSFLM [] and scaled with SCALA []. Crystals of the ADP complex were of space group P21 with cell dimensions a = 43.6 Å, b = 66.3 Å, c = 61.8 Å, β = 103.6°. Crystals of the ternary complexes were of space group P212121 with cell dimensions a = 63.9 Å, b = 70.1 Å, c = 79.3 Å (ADP•APS) and a = 63.8 Å, b = 69.4 Å, c = 79.4 Å (AMP-PNP•APS), respectively, whereas the crystals of the Cys556Ser mutant belonged to space group C2221 with cell dimensions a = 68.2 Å, b = 71.1 Å, c = 118.6 Å. Details of the data collection statistics are given in .The structure of CysC•ADP was determined by molecular replacement with PHASER [] using chain A of the human PAPS synthetase 1 (PAPSS1) structure (2PEYA) as search model []. Crystallographic refinement consisted of iterative rounds of refinement with REFMAC5 [] (Murshudov et al., 1997) and model building using COOT []. Local NCS restraints were used in the refinement of all structures.The crystal structures for both ternary complexes and the Cys556Ser mutant were determined by molecular replacement using the refined coordinates for the CysC•ADP complex as search model, with the ADP molecule removed from the model. Refinement followed the same procedure as described above, except that for the ternary complexes TLS refinement was included. Structure refinement was carried out by manual adjustments in COOT interspersed by minimization in Refmac5. Water molecules were added using COOT or PHENIX []. Structure validation was performed with MolProbity []. Structure figures were prepared with PyMOL and CCP4MG []. The atomic coordinates and structure factors amplitudes reported in this paper have been deposited in the Protein Data Bank with PDB ID codes: 4BZP, 4BZQ, 4BZX and 4RFV. […]

Pipeline specifications

Software tools iMosflm, CCP4, REFMAC5, Coot, MolProbity, PyMOL, CCP4mg
Applications Small-angle scattering, Protein structure analysis
Organisms Mycobacterium tuberculosis, Homo sapiens, Mycobacterium
Chemicals Adenosine Triphosphate, Ampicillin, Cysteine, Nucleotides