Computational protocol: FolX from Pseudomonas aeruginosa is octameric in both crystal and solution

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Protocol publication

[…] Crystals were flash-frozen in liquid nitrogen, with no further cryo-protection, and brought to Diamond Light Source, station I03. Data were collected on a PILATES 6M detector, at a wavelength of 0.97625 Å. A total of 127° of data were collected using an increment of 0.15°. Data were processed using MOSFLM and scaled in SCALA . The structure was determined using the Balbes molecular replacement server , which identified PDB entry 1B9L as the best search model. The solved structure was refined with BUSTER , using TLS parameterisation and torsion restraints from the search model, and inspected and altered when required, using COOT . Waters were added using BUSTER. The geometry of the structure was validated by MOLPROBITY . The data showed signs of anisotropy and were processed to 3.0 Å, based on the statistical factors presented in , although diffraction spots could be detected closer to 2.5 Å. [...] SAXS data were collected at the ESRF ID14 EH3 beamline for samples with a range of FolX concentrations of between 0.5 and 6 mg ml−1 in 20 mM Tris pH 7.5, 150 mM NaCl. No concentration dependence effects were observed, therefore data from the highest concentration (6 mg ml−1) were processed for further analysis. Initial processing of the data was done using PRIMUS and p(r) analysis was carried out using GNOM . The resolution of the data was calculated from the highest angle at which useable scattering data were recorded Ab initio models of FolX were generated from the experimental data using DAMMIF . Twenty DAMMIF models were superimposed and averaged using DAMAVER and the averaged model was superimposed onto the crystal structure using SUPCOMB .Figures were made using ALINE and PyMOL . […]

Pipeline specifications

Software tools iMosflm, CCP4, Coot, MolProbity, ATSAS, DAMMIF, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Pseudomonas aeruginosa, Dipturus trachyderma