Computational protocol: The structure of the cysteine protease and lectin-like domains of Cwp84, a surface layer-associated protein from Clostridium difficile

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Protocol publication

[…] Crystallization-condition screening was performed with a range of pre-prepared 96-well screens (Molecular Dimensions) using an Art Robbins Phoenix nanodispensing robot. Optimal conditions were reproduced with 0.3 µl drops with a 1:1 ratio of protein to reservoir solution (0.2 M ammonium sulfate, 30% PEG 4K; Molecular Dimensions Structure Screen 1 & 2, solution D7). Crystals took between 3 d and a week to grow.X-ray diffraction data were collected at station I03 at Diamond Light Source (DLS; Didcot, Oxfordshire, England). The diffraction data were recorded with 1.0° oscillation on a Pilatus 6M detector from four crystals to obtain maximum redundancy. Selenium-fluorescence peak and inflection data were collected from all four crystals (to a maximum resolution of 1.73–1.87 Å), while high and low remote data were collected from two crystals (to a maximum resolution of 1.94–2.16 Å). 1120 peak images were collected at 12 660 eV, 1120 inflection images at 12 656 eV, 540 low-remote images at 12 550 eV and 540 high-remote images at 12770 eV. The data were automatically indexed and integrated with XDS (Kabsch, 2010) and xia2 (Winter et al., 2013), respectively. The data were scaled (and resolutions cut to those reported in Table 1 to reduce errors) with SCALA (Diederichs & Karplus, 1997), combined with CAD (CCP4; Winn et al., 2011) and put into the Crank MAD pipeline (CCP4; Ness et al., 2004) with a resolution cutoff of 2.5 Å using SCALEIT (Howell & Smith, 1992), AFRO (CCP4), CRUNCH2 (de Graaff et al., 2001), BP3 (Pannu et al., 2003; Pannu & Read, 2004), SOLOMON (Abrahams & Leslie, 1996) and 500 cycles of Buccaneer/REFMAC (Cowtan, 2006; Murshudov et al., 2011). CRUNCH2 found 55 potential selenium sites out of a predicted 48 within the unit cell, the validity of which was determined with the later programs, allowing Buccaneer and REFMAC to produce an output model with a figure of merit of 85.6% and R cryst and R free values of 24.8 and 27.7%, respectively. The model was further refined with Coot/REFMAC (Emsley & Cowtan, 2004) using a 1.4 Å resolution native data set collected on a Pilatus 6M on I02 at DLS that had been autoprocessed with XDS and xia2 and scaled with AIMLESS (Evans, 2006). Secondary structure was determined using DSSP (Kabsch & Sander, 1983) and the model was verified with MolProbity (Chen et al., 2010).The atomic coordinates and structure-factor amplitudes have been deposited with the RCSB Protein Data Bank ( under PDB accession code 4ci7. […]

Pipeline specifications

Software tools Buccaneer, MolProbity
Applications Small-angle scattering, Protein structure analysis
Organisms Clostridioides difficile
Chemicals Amino Acids, Calcium, Cysteine