Computational protocol: Activity of acetyltransferase toxins involved in Salmonella persister formation during macrophage infection

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Protocol publication

[…] The purified TacT3Y143F proteins (40 mg/ml) crystallized by sitting drop vapour diffusion against a reservoir of 10% PEG 6000, 100 mM Bicine NaOH pH 9.0. Crystals were harvested and submerged in a crystallization buffer containing 20% glycerol for 10 s followed by freezing by immersion into liquid nitrogen. Diffraction data were acquired at diamond light source (Oxfordshire, UK) on beamline I04–1. High-resolution native data sets were auto-integrated using autoPROC software. Phases was obtained with PHENIX Phaser software using molecular replacement function and a polyalanine scaffold of TacT (PDB 5FVJ) as a model. The model was then modified using AutoBuild and manually improved using Coot and refined using PHENIX and Refmac, against the high-resolution native data to give a final structure with an Rfree of 23.1% and good geometry (Supplementary Table ). The relatively high final R factor could be explained by the flexible loop in the 75–82 region in one of the monomer units. […]

Pipeline specifications

Software tools PHENIX, Coot
Application Protein structure analysis
Organisms Escherichia coli, Homo sapiens, Salmonella enterica subsp. enterica serovar Enteritidis, Salmonella enterica subsp. enterica serovar Typhimurium
Diseases Salmonella Infections, Typhoid Fever