Computational protocol: Structure of Gentlyase, the neutral metalloprotease of Paenibacillus polymyxa

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Protocol publication

[…] For crystallization trials, the protein was concentrated to 10 mg ml−1. Screening was performed in sitting drops at 277 K and crystals grew from several conditions containing PEG. A crystal grown from 0.2 M NaCl, 0.1 M HEPES pH 7.5, 25% PEG 3350 (Index condition F12) was soaked with the inhibitor phosphoramidon at 10 mM and a crystal grown from 0.2 M MgCl2, 0.1 M HEPES pH 7.5, 25% PEG 3350 (Index condition G12) was harvested directly. Both crystals were flash-cooled in liquid nitrogen without further addition of cryoprotectant. Data collection was performed at 100 K on beamline XS10A at the Swiss Light Source. The wavelength was 1 Å. Images were processed with XDS (Kabsch, 2010) and scaled with SADABS (Bruker AXS) or XDS. The structure was solved by molecular replacement with Phaser (McCoy et al., 2007) using thermolysin (PDB entry 3fvp; L. Englert, A. Biela, M. Zayed, D. Hangauer, A. Heine & G. Klebe, unpublished work) as a search model. It was rebuilt with ARP/wARP (Perrakis et al., 1999) and refined with REFMAC (Murshudov et al., 2011); further structural modelling was performed with Coot (Emsley & Cowtan, 2004). The CCP4 program suite was used throughout (Winn et al., 2011). The Ramachandran quality of the structural models was calculated by Coot to be 96.8/3.2/0.0 for the peptide complex and 93.3/6.5/0.2 for the phosphoramidon complex, where the values reflect the percentage of amino-acid residues in the core, allowed and disallowed regions of the Ramachandran plot, respectively. Data-collection and refinement statistics are given in Table 1. […]

Pipeline specifications

Software tools XDS, ARP/wARP, Coot, CCP4
Applications Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma, Paenibacillus polymyxa, Bacillus cereus
Chemicals Calcium