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Protocol publication

[…] The American Type Culture Collection (ATCC) provided the genomic DNA used to clone ipi (ATCC Number: 23857D-5). Protein production and crystallization of IPI was carried out by standard JCSG protocols . The crystal was obtained using the vapor diffusion method in a sitting drop format where sitting drops composed of 100 nl protein solution mixed with 100 nl crystallization solution were equilibrated against a 50 μl reservoir at 293 K. The crystallization reagent consisted of 48.5% polyethylene glycol 600, 0.1M CHES pH 9.7. Data were collected at wavelengths corresponding to the inflection and high energy remote of a selenium MAD (multi-wavelength anomalous dispersion) experiment at 100 K using a MARCCD 325 detector (Rayonix) at Stanford Synchrotron Radiation Lightsource (SSRL) beamline 9_2. Data processing were carried out using XDS and the statistics are presented in . The structure was determined by the MAD method using programs SHELX and autoSHARP , and refinement was carried out using REFMAC5 . The structure was validated using the JCSG Quality Control server ( http://smb.slac.stanford.edu/jcsg/QC). Atomic coordinates and experimental structure factors to 2.6 Å resolution (PDB code: 3ISY) have been deposited in the Protein Data Bank ( www.pdb.org ). [...] Sequence conservation among homologues of IPI was mapped onto the crystal structure using ConSurf ( http://consurf.tau.ac.il/; ). The results were visualised with PyMOL ( http://www.pymol.org/), which was also used for structural figures.A homology model of B. subtilis intracellular proteinase, IspA, was created at the Swiss-model server using the structure of processed, active intracellular protease from Bacillus clausii (PDB code 2XRM), around 50% identical to IspA, as the sole template. The high degree of sequence identity with the target and the small number of insertions and deletions between target and template (one six residue deletion and a single insertion of one residue, both readily accommodated) assured a high quality model. Additionally, the insertions and deletions lie distant from the catalytic site which was the main region of interest.The new structure of IPI was docked to the model of IspA using three different webservers, GRAMM-X ( http://vakser.bioinformatics.ku.edu/resources/gramm/grammx/; ), ZDOCK ( http://zdock.umassmed.edu/; ) and ClusPro 2.0 ( http://cluspro.bu.edu/; ). […]

Pipeline specifications

Software tools ConSurf, PyMOL, GRAMM-X, ZDOCK, Cluspro
Application Protein interaction analysis
Organisms Bacillus subtilis
Chemicals Cysteine