Computational protocol: Structural Basis of Catalysis in the Bacterial MonoterpeneSynthases Linalool Synthase and 1,8-Cineole Synthase

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Protocol publication

[…] All data were collected at Diamond Light Source (DLS). Diffraction images were integrated and scaled by xia2 automated data processing pipeline, using XDS and XSCALE. Crystals of bCinS contained two molecules in the asymmetrical unit and belonged to P1 space group. Crystals of bLinS belonged to the tetragonal system (spacegroup I4) and also contained two molecules in asymmetrical unit. The bLinS structures (apo-bLinS and bLinS-FGPP) were solved by molecular replacement using the Pentalenene synthase structure (PDB: 1PS1()) as the search model in Phaser. The bCinS-FNPP structure was solved by model replacement using the apo-bLinS structure as the search model. The apo-bLinS, bLinS-FGPP, bCinS-FNPP and bCinS-FNPP/FGPP models were built using Autobuild in Phenix. The structures were completed using iterative rounds of manual model building in coot and refinement in phenix.refine. The structures were analyzed using PDB_REDO and validated using molprobity tools. The refinement statistics are provided in . The atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 5NX4, 5NX5, 5NX6 and 5NX7. [...] Molecular dynamics (MD) simulations of apo-bCinS and bLinS were carried out in AMBER14 using the CHARMM27 force field., The protonation states of titratable residues were estimated using the PDB 2PQR server with proPKA, and the enzymes were solvated using a box of minimum 12 Å around the protein with counterions added. Two sets of isothermal–isobaric ensemble (NPT) MD simulations were performed at 298 K for each enzyme, using different starting velocities, following the system setup. Langevin dynamics was used for temperature control (collision frequency of 5 ps–1 for equilibration and 2 ps–1 for production), and pressure was controlled by coupling to an external bath (AMBER14 default settings) for NPT conditions. The system setup consisted of: (i) energy minimization of the solvent; (ii) 50 ps of (NPT) solvent equilibration; (iii) energy minimization of the entire system with positional restraints of 5 kcal mol–1 Å–2 applied to all Cα atoms; (iv) canonical ensemble (NVT) thermalisation to 298 K over 20 ps with positional restraints of 5 kcal mol–1 Å–2 on Cα atoms; (v) 40 ps of NPT equilibration with decreasing restraints on the Cα atoms; (vi) 1 ns unconstrained NPT equilibration; (vii) 100 ns production simulation. Average linkage hierarchical clustering (after alignment of structures based on Cα positions) was used to identify representative structures to illustrate protein conformational sampling during the simulations. […]

Pipeline specifications

Software tools xia2, XDS, PHENIX, Coot, MolProbity, AMBER, PROPKA
Databases PDB_REDO
Applications Small-angle scattering, Protein structure analysis
Organisms Bacteria, Streptomyces clavuligerus, Escherichia coli