Computational protocol: Towards new cholera prophylactics and treatment: Crystal structures of bacterial enterotoxins in complex with GM1 mimics

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[…] Samples were mounted in loops, flash-frozen in a nitrogen cryo-stream, and subjected to data collection at the ESRF, Grenoble, France– (Table ). Scaling and processing of the CTB data sets was done with XDS, whereas the pLTB data set was scaled and processed with Mosflm . Diffraction cut-offs were chosen based on the assessment of CC1/2 , (Table ), and the quality of the electron density. The pLTB + 3 diffraction data were collected using remote access, to a resolution of 1.60 Å, which in hindsight turned out to be a very conservative cut-off with CC1/2 = 90.2. The structures were solved by molecular replacement using MOLREP from the CCP4 software suite. The search model used for the CTB structures was a 1.25 Å crystal structure of cCTB (PDB ID: 3CHB). The pLTB R13H structure was solved using the native pLTB crystal structure (PDB ID: 1EFI) as a search model. The search models were prepared by pruning unconserved residues and removing water molecules with the program CHAINSAW . The inhibitors were modelled in MarvinSketch and MarvinSpace (, and the corresponding PDB- and library files were created using PRODRG . After initial rigid body refinement using REFMAC5 , setting 5% of the reflections aside for calculating the R free, the structures were surveyed and patched with Coot and further refined. At later stages of the refinement, water molecules were manually added with Coot. The inhibitors were included last. All inhibitors were modelled with 100% occupancy after assessment of the difference electron density and B-factors of the ligand and the nearby protein chain.The data sets with inhibitor 2 showed significant anisotropy, which resulted in disproportionally high R-factors, a common effect of anisotropy. Although the Hamilton R ratio test showed that the structures could also be refined with anisotropic B-factors, this was abandoned in favour of an isotropic B-factor model with TLS refinement, since anisotropic B-factors resulted in inferior electron density in the loop areas (residues 50–60), and made model building harder. The application of TLS refinement resulted in well-defined electron density also in the loop areas, along with R/R free values comparable to those obtained from anisotropic B-factor refinement. In the structure of cCTB in complex with 2 some of the intramolecular disulphide bridges (Cys9-Cys86) show oxidation, suggesting radiation damage. Cys9 has previously been reported to have two conformations, one of them pointing away from Cys86 and Thr15, . However, in this structure, strong positive difference density (10 r.m.s.d.) was observed on the opposite site of the disulphide link, towards Thr15, indicating a modification of the sulphur atom rather than an alternative conformation of the residue. Partial oxidation of the residues was modelled by replacing the cysteines with S-oxy cysteine (CSX), with 50% occupancy of the oxygen in chains A, B and D.The pLTB R13H + 3 data set is of high quality with close to 100% completeness, while the ET CTB + 1 data set exhibited some anisotropy. Both structures were refined with standard isotropic B-factors. […]

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