Computational protocol: Structure and Mechanism of Human UDP-xylose Synthase

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Protocol publication

[…] SeMet-labeled hUXS1 containing 5 mm NAD+ and 5 mm UDP was concentrated to 12 mg/ml (∼0.3 mm). Crystals were obtained by vapor diffusion at 20 °C in 150-nl sitting drops, equilibrated against mother liquor containing 20% PEG 6000, 10% ethylene glycol, and 0.2 m NaCl in 0.1 m HEPES (pH 7). Diffraction data to 1.2 Å were collected at 100 K at the Swiss Light Source station X10SA using a MARMOSAIC 225 mm CCD detector at a single wavelength near the selenium absorption edge (λ = 0.9795 Å). Data were indexed and integrated in MOSFLM () and scaled in SCALA (). Merging of the data and analysis of systematic absences identified the space group as P121, with the following cell dimensions: a = 46 Å, b = 45 Å, c = 85 Å; α = 90°, β = 97°, γ = 90°. The program SHELXD () located 11 SeMet residues in hUXS1, indicating complete labeling of the protein. SHELXE was used for phasing and automated chain tracing was done with ARP/wARP. The hUXS1 structure was manually rebuilt in COOT (), and restrained refinement was performed using REFMAC5 (). When electron density showed the presence of NAD+ and UDP in the active site as well as an additional UDP forming a crystal contact, the ligands were fitted to the density and included in the refinement. Although the full range of data to 1.2 Å was used in refinement, analysis of data completeness and signal to noise suggests that 1.26 Å is a more reasonable estimate of the resolution of the model. Data collection and refinement statistics are summarized in . [...] All MD simulations were performed using Gromacs (). For the protein and water a variation of the Amber99sb () and the TIP3P () force fields was used. For NAD, substrate, intermediates, and product General Amber Force Field (GAFF) parameters () and Gromacs input files were generated using the Amber Antechamber suite of programs () together with the perl script amb2gmx (). As starting structure for MD simulations the crystal structure of UXS (PDB code 2B69) was used, approximate pKa values of ionizable residues were established using PROPKA (), and ionizable residues were protonated accordingly. For the MD simulations the protein with substrate, product, or intermediates was positioned in the center of a cubic box with 6.7-nm side length and solvated in ∼8,000 water molecules. The appropriate number of water molecules was replaced by sodium ions to neutralize the system. A leapfrog algorithm with a time step of 2 fs was used for integrating the equations of motion. The temperature was kept constant at 300 K using a Nosé-Hoover thermostat (). Electrostatic long range interactions were accounted for using a particle mesh Ewald algorithm (). A cut-off of 10 Å was used for the real space part of the Ewald summation and the van der Waals interactions. A constant pressure of 1 atm was maintained using a Berendsen thermostat (). All bonds including hydrogens were constrained using a LINCS algorithm (). Conformations were saved every 2 ps. The MD simulations covered a time between 4 and 6 ns. In each case the trajectories covering the last 2 ns were used to calculate hydrogen bonding and average structures. For visual inspection of trajectories and conformations Chimera was used. The trajectories were analyzed with various Gromacs tools and in-house scripts. […]

Pipeline specifications

Software tools iMosflm, CCP4, SHELX, ARP/wARP, Coot, REFMAC5, GROMACS, PROPKA, P-LINCS
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens
Chemicals Glycosaminoglycans, NAD, Pentosephosphates, Uridine Diphosphate, Uridine Diphosphate Glucuronic Acid, Uridine Diphosphate Xylose, Hydroxyl Radical