Computational protocol: Crystal Structure and Pyridoxal 5-Phosphate Binding Property of Lysine Decarboxylase from Selenomonas ruminantium

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Protocol publication

[…] The crystals were transferred to a cryo-protectant solution composed of the corresponding condition described above and 30% (v/v) glycerol, fished out with a loop larger than the crystals, and flash-frozen by immersion in liquid nitrogen. All data were collected at the 7A beamline of the Pohang Accelerator Laboratory (PAL, Pohang, Korea), using a Quantum 270 CCD detector (ADSC, USA). The crystals of the apo-forms I and II of SrLDC diffracted to 2.0 and 2.9 Å resolutions, respectively. The crystals of SrLDC complexed with PLP/cadaverine crystals diffracted to 2.5 Å resolutions. All data were indexed, integrated, and scaled using the HKL-2000 software package []. The apo-form I crystals of SrLDC belonged to the space group P43212 with unit cell parameters a = b = 105.97 Å, c = 73.634 Å, α = β = γ = 90.0°. With one molecule of SrLDC per asymmetric unit, the crystal volume per unit of protein mass was 2.35 Å3 Da-1, indicating that the solvent content was approximately 47.63% []. The apo-form II of SrLDC crystals belonged to the space group C2 with unit cell dimensions of a = 146.45 Å, b = 70.794 Å, c = 88.061 Å, α = γ = 90.0°, β = 101.03°. With two molecules per asymmetric unit, the crystal volume per unit of protein mass was 2.54 Å3 Da-1, which corresponds to a solvent content of 51.68% []. The PLP/cadaverine-complexed form of SrLDC crystals belonged to the space group P6322 with unit cell dimensions of a = b = 111.73 Å, c = 112.97 Å, α = β = 90.0°, γ = 120.0°. With one molecule per asymmetric unit, the crystal volume per unit of protein mass was 2.31 Å3 Da-1, and the solvent content was 46.83% []. The structure of the apo-form I of SrLDC was solved by molecular replacement with the CCP4 version of MOLREP [] using the structure of L/ODC from Vibrio vulnificus (VvL/ODC, PDB code 2PLK) as a search model. Model building was performed manually using the program WinCoot [] and refinement was performed with CCP4 refmac5 []. The structures of the apo-form II of SrLDC, SrLDC complexed with PLP/cadaverine were determined by molecular replacement using the refined SrLDC structure of apo-form I. Model building and structure refinement of these structures were performed using a procedure similar to that employed for the structure of the apo-form I of SrLDC. Data collection and refinement statistics are summarized in . Four refined SrLDC structures of apo-from I, apo-form II, and complexed with PLP/cadaverine were deposited in the Protein Data Bank with PDB codes of 5GJN, 5GJM, and 5GJP, respectively. […]

Pipeline specifications

Software tools HKL-2000, CCP4, Molrep, REFMAC5
Application Protein structure analysis
Organisms Selenomonas ruminantium
Chemicals Cadaverine, Pyridoxal Phosphate