Computational protocol: Structure of a Switchable Subtilisin Complexed with a Substrate and with the Activator Azide†

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Protocol publication

[…] The s164−pG16 complex was crystallized from 15% (v/v) PEG 8000, 0.2 M zinc acetate, and 0.1 M sodium cacodylate (pH 6.2) as previously described (). For the anion-free complex, a crystal was cryoprotected by immersion for 2 s in 7% glycerol and then cryocooled by immersion in liquid nitrogen. The fluoride-soaked crystal was immersed for 2 min in reservoir solution to which 200 mM KF had been added and then cryoprotected and frozen. The azide complex crystal was immersed for 3 min in reservoir solution to which 25 mM sodium azide had been added, prior to cryoprotection and freezing. Diffraction data for the anion-free crystal were collected on beamline 24ID of the Argonne Synchrotron, while data for the other two crystals were collected on a rotating anode R-axis IV diffractometer. The anion-free structure was determined by molecular replacement as described previously (). Diffraction data sets to resolutions of 1.7, 1.9, and 1.8 Å were collected for the three structures, 3CNQ, 3CO0, and 3BGO, respectively. Refinements of all three structures using REFMAC5 () and XFIT () led to Rfree values of 0.23, 0.29, and 0.26, respectively. For each of the three structures, a difference map showing the scissile and C-terminal tail regions of the substrate, as well as the side chain of His 64 and the contents of the anion-binding pocket, was calculated before these components had been added to the structure, but after the remainder of the structure had been refined. These key components were thus built into unbiased maps. The molecular graphics program Coot () was used to superpose the three structures. Coordinates and complete statistics are available from the RCSB Protein Data Bank as entries 3CNQ (anion-free), 3CO0 (fluoride-soaked), and 3BGO (azide-bound). […]

Pipeline specifications

Software tools REFMAC5, Coot
Application Protein structure analysis
Organisms Bacillus amyloliquefaciens
Chemicals Fluorides