Computational protocol: High resolution crystal structures of the receptor-binding domain of Clostridium botulinum neurotoxin serotypes A and FA

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Protocol publication

[…] Crystals of His6-HC/FA and His6-HC/A1 were grown at 16 °C using a 1:1 ratio of protein solution (10 mg/mL) to well solution using the sitting-drop vapour-diffusion method—4 M sodium formate, 0.1 M sodium acetate pH 5.5 for the former, and 0.1 M MIB pH 4.0, 25% w/v PEG 1500 for the latter. Crystals were soaked in cryoprotectant (equal volume of reservoir solution and 50% glycerol) before vitrification in liquid nitrogen. Complete datasets were collected on beamline I03 and I04, respectively, at the Diamond Light Source (Didcot, UK). Diffraction images were processed using DIALS () and scaled using AIMLESS () from the CCP4 suite (). Data collection statistics are summarised in . A combination of Rpim and CC1/2 value were used to determine the resolution cut-off of 1.95 Å and 1.45 Å, respectively. Phase information was determined by molecular replacement using PHASER () and a previous structure of HC/A1 (PDB: 2VUA; ) as the initial search model. Multiple rounds of structure refinement were performed by manual correction in COOT () followed by restrained refinement with REFMAC5 (). Final validation was performed with MolProbity (). Secondary structures were annotated using Stride () and figures were prepared using PyMol (Schrödinger, LLC, New York, NY, USA) and CCP4mg (). […]

Pipeline specifications

Software tools CCP4, Coot, REFMAC5, MolProbity, STRIDE, PyMOL, CCP4mg
Application Protein structure analysis
Organisms Clostridium botulinum