Computational protocol: Purification, crystallization and X-ray structures of the two manganese superoxide dismutases from Caenorhabditis elegans

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Protocol publication

[…] Data for MnSOD-3 were recorded at 293 K with an R-AXIS IV++ image-plate detector mounted on a Rigaku RU-H3R rotating-anode X-ray generator and were integrated using MOSFLM (Leslie, 1999) to 1.77 Å resolution. Subsequently, X-ray diffraction data for both MnSOD-2 and MnSOD-3 were recorded at 100 K on station I03 at Diamond Light Source using an ADSC Quantum 315 charge-coupled device (CCD) detector. The Diamond Light Source MnSOD-2 and MnSOD-3 data were integrated to 1.8 and 1.7 Å resolution, respectively, using the program MOSFLM. Data reduction and subsequent calculations were carried out using the CCP4 program suite (Collaborative Computational Project, Number 4, 1994). Both MnSOD-2 and MnSOD-3 crystals belonged to space group P41212, with unit-cell parameters a = b = 81.0, c = 137.4 Å for MnSOD-2 and a = b = 81.8, c = 136.0 Å for MnSOD-3. There are two subunits of MnSOD-2 and MnSOD-3 per asymmetric unit (Table 1). [...] The crystal structure of MnSOD-3 solved at 293 K was determined by molecular replacement using the program Phaser (Read, 2001) with the human MnSOD enzyme structure (PDB code 1n0j; Borgstahl et al., 1992) as the search model (63.6% sequence identity). After initial rounds of rigid-body and restrained refinement using REFMAC5 (Murshudov et al., 1997), the polypeptide chain was mutated to the MnSOD-3 residues and checked against both 2F o − F c and F o − F c maps using the program Coot (Emsley & Cowtan, 2004). There were two deletion regions compared with the human model: one that was missing Gly87 and the other Gly147, Thr148 and Thr149 (Fig. 1). Residues around these regions that did not fit well to the map were removed (85–91 and 147–151; MnSOD-3 numbering) to prevent bias in map generation. Model building and refinement were carried out using Coot and REFMAC5, allowing the deleted regions to be rebuilt.The structure of MnSOD-3 solved at 100 K was determined at 1.7 Å resolution from the 293 K MnSOD-3 structure by difference Fourier. Model building and refinement were carried out using Coot and REFMAC5, allowing 420 water molecules and one malonate ion to be rebuilt, with the R free set carried over from the 293 K structure. Noncrystallographic symmetry averaging was not used during structural refinement. In the later stages of refinement, TLS parameters (Winn et al., 2001) based on a single-group TLS model for each monomer were calculated using the TLS Motion Determination server (http://skuld.bmsc.washington.edu/~tlsmd/) and refined in REFMAC5. The refined models of MnSOD-3 at both 293 and 100 K each contain two protein chains in the asymmetric unit, comprising all amino acids in the sequence. The final structure of MnSOD-3 was refined to R = 21.6% and R free = 26.2% at 293 K and R = 18.9% and R free = 22.6% at 100 K.The structure of MnSOD-2 was determined at 1.8 Å resolution from the 100 K MnSOD-3 structure by difference Fourier. Model building and refinement were carried out using Coot and REFMAC5. Noncrystallographic symmetry averaging was not used during structural refinement. TLS parameters based on a single-group TLS model for each monomer (calculated from the TLS Motion Determination server) were refined at later stages of refinement. The final structure of MnSOD-2 was refined to R = 16.9% and R free = 20.1%. […]

Pipeline specifications

Software tools SHELX, iMosflm, CCP4, REFMAC5, Coot
Applications Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma, Caenorhabditis elegans, Mus musculus, Homo sapiens