Computational protocol: Structural Basis of Substrate Binding in WsaF, a Rhamnosyltransferase from Geobacillus stearothermophilus

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Protocol publication

[…] X-ray diffraction data from a single crystal of native WsaF were collected to a resolution of 3.0 Å on the ID14-4 beamline (λ = 0.979) at the European Synchrotron Radiation Facility (Grenoble, France) equipped with an ADSC Q315 detector. Since no suitable model for molecular replacement was available, we used a SeMet-labelled derivative of WsaF to solve the structure with experimental phases. Highly redundant X-ray diffraction data (redundancy of 14.4) from a single P21 crystal of SeMet-labelled WsaF were collected to 2.28 Å resolution on the I03 beamline (λ = 0.9764) at Diamond (Didcot, UK) equipped with an ADSC Q315 detector. The data were processed and scaled with HKL2000. PHENIX.HYSS found 10 of the 16 SeMet residues in the asymmetric unit (two WsaF molecules). PHENIX.AUTOSOLVE was used to phase the data and to build an initial model of WsaF. The initial model was refined with PHENIX.REFINE and then manually improved in the molecular graphics program Coot. Subsequent refinement was carried out with REFMAC5 and TLS groups employed. Statistics are shown in . Difficulty in reproducing the crystallisation experiments prompted us to switch to WsaFSER2 for further structural studies. Data sets of the WsaFSER2-dTDP and WsaFSER2-dTDP-β-l-Rha complexes were collected in-house using Cu-Kα radiation on a Rigaku Saturn 944 detector (λ = 1.5418). The data were processed and scaled with XDS, and the structures were solved by molecular replacement using Phaser with the structure of the apoenzyme as search model. The dTDP and dTDP-β-l-Rha ligands were located in the initial difference Fourier maps and manually positioned using Coot. The ligands have higher B-factors than the protein, reflecting less than full occupancy. We felt that the resolution was insufficient to accurately model both B-factor and occupancy in the model; thus, occupancy was set to 1. Refinement was performed using REFMAC5 and PHENIX. […]

Pipeline specifications

Software tools PHENIX, Coot, REFMAC5
Applications Small-angle scattering, Protein structure analysis
Organisms Geobacillus stearothermophilus