Computational protocol: Biochemical and structural characterisation of the second oxidative crosslinking step during the biosynthesis of the glycopeptide antibiotic A47934

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Protocol publication

[…] Crystals were grown using hanging drop vapour diffusion at 4 °C. The StaF protein (140 µM) was mixed (1:1) with the reservoir solution (0.1 M phosphate/citrate buffer (pH 4.2), 1.2 M Na2PO4, 0.3 M K2HPO4; final pH 5.2) and equilibrated against the reservoir solution. After 10 days red diamonds (≈150 µm length) had formed. The crystals were passed through a cryoprotectant solution (0.1 M phosphate/citrate buffer (pH 4.2), 1.2 M Na2PO4, 0.3 M K2HPO4 and either 25% (v/v) glycerol, or 25% (v/v) ethylene glycol) and then flash cooled in liquid nitrogen for data collection. Two native data sets using different cryoprotectant solutions were collected at the X10SA beamline at the Swiss Light Source at the Paul Scherrer Institute (Villigen, Switzerland, λ = 0.9792 Å) with the crystals kept at 100 K during data collection. The data was processed using the XDS program suite []. The space group of the crystals was P3(1)2(1) with a single P450 molecule per asymmetric unit. The StaF structure was solved using molecular replacement with the program PHASER [] and a search model consisting of OxyEtei (Protein Data Bank code 3O1A, Chain A) [], residues 2-384 and heme. Iterative manual model building and refinement were performed using the programs COOT [] and REFMAC [] with TLS refinement [] following a simulated annealing performed in CNS [–]. During several rounds of refinement with REFMAC and manual rebuilding, ethylene glycol or glycerol and solvent molecules were included in the models where appropriate. TLS input files were generated using the TLS-Motion Determination Server [–]. Structure validation was performed using MOLPROBITY [] and PROCHECK []. Structure-based sequence alignments were carried out with SSM [] as implemented in COOT and comparisons to known structures performed with DaliLite []. All structural figures were prepared using PyMol []. Atomic coordinates and structure factor amplitudes have been deposited in the Protein Data Bank (PDB) under accession codes 5EX8 (ethylene glycol cryoprotectant solution) and 5EX9 (glycerol cryoprotectant solution). […]

Pipeline specifications

Software tools XDS, Coot, CNS, MolProbity, PROCHECK, DALI, PyMOL
Applications Small-angle scattering, Protein structure analysis
Chemicals Amino Acids