Computational protocol: Biochemical Discrimination between Selenium and Sulfur 1: A Single Residue Provides Selenium Specificity to Human Selenocysteine Lyase

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Protocol publication

[…] Initial crystallization conditions were identified from the JCSG+ crystal screen (QIAGEN). Crystals were grown from sitting drops containing 0.1 µl of protein solution (17 mg/ml)+0.1 µl well solution having 100 mM HEPES pH 6.7 and 10% PEG6000 that were left to equilibrate against the well solution. Crystals were grown at 20°C and appeared after three days. Data was collected at the ESRF, beam-line ID23-1 at λ = 1.071 Å and 100 K. Data was processed with XDS and XSCALE . The space group was P212121 with cell parameters a = 59.2 Å b = 86.8 Å c = 189.6 Å and one protein dimer in the asymmetric unit. The structure was solved by Molecular Replacement using a monomer of the E. coli cysteine desulfurase IscS (pdb entry: 1P3W) as the search model. Refmac was used for refinement and Coot for model building. TLS refinement with 4 TLS groups was used in Refmac. The final model starts at Glu29 and ends at Gln444, the penultimate residue in the sequence. Residues 120 to 132 in both monomers are disordered and could not be detected in the electron density. 92.3% of the residues are in the favored region, 7.3% in the allowed, 0.4% in the generously allowed and no residues in the disallowed region of the Ramachandran plot calculated using PROCHECK . In addition, diffraction data was also collected from crystals grown from sitting drops containing 0.1 µl of protein solution (17 mg/ml) with 0.1 µl well solution containing 50 mM HEPES pH 8.1, 200 mM Ammonium Nitrate and 25% PEG3350 that were left to equilibrate against the well solution. In this case, crystals appeared after seven days at 20°C. Crystals were incubated in well solution supplemented with 10 mM L-cysteine for 2 hours. Data was collected at the ESRF, beam-line BM14 at λ = 0.9800 Å and 100 K. Data was processed with XDS and XSCALE . The space group was P1 with cell parameters a = 66.6 Å, b = 72.2 Å, c = 89.4 Å, α = 83.9° β = 68.4°, γ = 87.0°. The structure was solved by Molecular Replacement using a monomer of the P212121 structure as the search model. The asymmetric unit contained two protein dimers. Refmac was used for refinement and Coot for model building. The final model starts at Lys31 and ends at Gln444. Residues 120 to 132 were disordered and invisible in the electron density, which in this case was also the case for the active site loop residues 391 to 394. 92.7% of the residues are in the favored region, 7.3% in the allowed and no residues in the generously or disallowed regions of the Ramachandran plot calculated using PROCHECK . Coordinates and structure factors have been deposited in the PDB with accession numbers 3GZC and 3GZD. […]

Pipeline specifications

Software tools XDS, Coot, PROCHECK
Applications Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma, Homo sapiens
Diseases Hamartoma Syndrome, Multiple
Chemicals Cysteine, Selenium, Sulfur, Selenocysteine