Computational protocol: Structural basis for redox sensitivity in Corynebacterium glutamicum diaminopimelate epimerase: an enzyme involved in l-lysine biosynthesis

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Protocol publication

[…] The crystals were transferred to cryoprotectant solution composed of the corresponding conditions 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 7 A beamline of the Pohang Accelerator Laboratory (PAL, Pohang, Korea), using a Quantum 270 CCD detector (ADSC, USA). The oxidized form and reduced form of CgDapF crystals diffracted to a resolution 2.3 and 2.0 Å, respectively. The crystals of CgDapF in complex with D,L-DAP diffracted to 2.6 Å resolutions. All data were indexed, integrated, and scaled together using the HKL-2000 software package. The oxidized form of CgDapF belonged to the space group P32 with the unit cell parameters a = b = 143.584 Å, c = 122.371 Å, α = β = 90.0°, and γ = 120.0°. In order to solve the phase problem for the crystal, we also prepared crystals from a SeMet derivative. SeMet-substituted oxidized form crystals were obtained using the same crystallization condition as used for the native protein crystal. The crystals of SeMet derivative diffracted to a resolution 2.0 Å and belonged to the space group I222 with the unit cell parameters a = 101.74 Å, b = 119.08 Å, c = 155.59 Å, and α = β = γ = 90°. Assuming two molecules of oxidized form of CgDapF (29.2 kDa) per asymmetric unit, the crystal volume per unit of protein mass was approximately 3.94 Å3 Da−1, which corresponded to a solvent content of 68.80%. The reduced form of CgDapF crystals that substituted with SeMet belonged to the space group I222 with unit cell parameters a = 101.76, b = 118.94 Å, c = 153.33 Å, α = β = γ = 90.0°. With two molecules of CgDapF per asymmetric unit, the crystal volume per unit of protein mass was 3.88 Å3 Da−1, which corresponded to a solvent content of 68.31%. The crystals of CgDapF in complex with D,L-DAP belonged to space group P4332, with unit cell parameters of parameters a = b = c = 155.7 Å, and α = β = γ = 90.0°. With one molecule of CgDapF in complex with D,L-DAP in an asymmetric unit, the crystal volume per unit of protein mass was 2.63 Å3 Da−1, indicating that the solvent content was approximately 53.26%. The crystal structures of SeMet-substituted CgDapF in oxidized and reduced form were solved by single-wavelength anomalous dispersion (SAD) using data collected at a determined wavelength (0.97934 Å). Experimentally determined f” and f”’ values by using SOLVE/RESOLVE. The initial model build was automatically performed using ARP/wARP, and the final model was built by using the program Wincoot. The structure of CgDapF in complex with D,L-DAP was determined by molecular replacement with the CCP4 version of MOLREP using the refined CgDapF in reduced form structure. Model building was performed manually using the program WinCoot, and refinement was performed with CCP4 refmac5. The data statistics are summarized in . The refined models of oxidized and reduced form of CgDapF and CgDapF in complex with D,L-DAP were deposited in the Protein Data Bank with PDB codes of 5H2G, 5H2Y, and 5M47, respectively. […]

Pipeline specifications

Software tools HKL-2000, ARP/wARP, Molrep, REFMAC5
Application Protein structure analysis
Organisms Corynebacterium glutamicum
Chemicals Cysteine