Computational protocol: Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexate

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Protocol publication

[…] A ternary complex of TbPTR1 with cofactor and MTX was prepared by incubating PTR1 (6 mg ml−1), 1 mM NADP+, 1 mM MTX and 20 mM dithiothreitol, all in 20 mM Tris-HCl pH 7.0, on ice for 20 min and crystallization screens carried out. Hanging drops were assembled by mixing 1.5 μl of protein solution with 1.5 μl of reservoir and incubated over 100 μl of reservoir, 0.1 M sodium cacodylate pH 6.5 and 1.4 M sodium acetate. Well-ordered monoclinic blocks (0.1 mm × 0.1 mm × 0.05 mm) grew at room temperature in several days.A crystal was briefly soaked in 30% glycerol and 70% of the reservoir solution then flashed cooled to −173°C in a stream of nitrogen gas (X-stream, Rigaku-MSC). Diffraction data were measured using a Rigaku Micromax 007 rotating anode (CuKα, λ = 1.5418 Å, 40 kV, 18 mA) and R-AXIS IV++ dual image plate detector system. Data to 2.2 Å resolution were collected using oscillations of 0.5° with an exposure time of 10 min per image, and processed using Denzo/Scalepack () and CCP4 () software. Five per cent of the data were flagged for the calculation of Rfree to monitor refinement protocols. The crystals display space group P21, with unit cell dimensions a = 74.6, b = 90.2, c = 80.8 Å, β = 115.8°. A homotetramer of total mass approximately 114 kDa constitutes the asymmetric unit.A poly Ala model for a subunit based on LmPTR1 (; Protein Data Bank code 1E92) was used in molecular replacement calculations (molrep; ). Four copies of this model, denoted subunits A–D, were positioned in the TbPTR1 unit cell with the quaternary structure typical of many SDR family members. Following rigid body refinement (refmac5; ), the Rwork was 40.1% (Rfree 46.9%) and the correlation coefficient was 0.56. Rounds of restrained maximum likelihood refinement, model manipulation and graphic inspection of electron density (2Fo-Fc) and difference density (Fo-Fc) maps (Fo is the observed structure-factor amplitudes, Fc the structure-factor amplitudes calculated from the model) were carried out using refmac5 and coot (). The placement of ligands, water molecules and assignment of several multiple conformers completed the analysis. NCS restraints were not imposed on the model during refinement.Several residues could not be modelled satisfactorily due to diffuse electron density. This applies to the surface loops that link β4 with α4, and α4 with β5. The residues in the first segment could not be identified in any of the four polypeptide chains of the asymmetric unit, and those from the latter segment could be modelled in subunit C only. Large positive features observed in difference density maps in the vicinity of Cys59 and Cys168, for all subunits, were compatible with covalent modification by cacodylate to form dimethylarsinoyl cysteine. Two positive difference density peaks were also observed between the His179 side-chains of subunits A and C, and chains B and D. These were modelled as Ni2+ and assigned occupancy of one-third. The presence of cacodylate and Ni2+ are artefacts of the crystallization and purification processes respectively. […]

Pipeline specifications

Software tools CCP4, Molrep, REFMAC5, Coot
Application Protein structure analysis
Organisms Trypanosoma brucei, Leishmania major
Chemicals Cysteine, Methotrexate, NADP