Computational protocol: Purification, crystallization and preliminary X-ray study of the fungal laccase from Cerrena maxima

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Protocol publication

[…] The crystal structure was solved by the molecular-replacement technique using the program MOLREP (Vaguine et al., 1999) from the CCP4 program suite (Collaborative Computational Project, Number 4, 1994). The structure of the laccase from Trametes versicolor (PDB code 1kya; Bertrand et al., 2002) was used as the search model. Only one solution was evident, with an R factor of 0. 413 and a correlation coefficient R corr of 0.642. The initial steps of refinement were performed using REFMAC (Murshudov et al., 1999). After a few cycles of rigid-body refinement, R and R free decreased to 0.368 and 0.382, respectively. Further restrained refinement decreased R and R free to 0.275 and 0.331, respectively.Several cycles of refinement with the CNS program suite (Brünger et al., 1998) were then performed using the Anneal, Minimize, Bgroup and Bindividual options. Stages of CNS refinement were alternated with manual correction of the model and the stereochemical parameters using the program O (Jones et al., 1991) and difference electron-density maps with (F o − F c), (2F o − F c) and (3F o − 2F c) coefficients. The final stages of model building involved the adjustment of the Cu-ion occupancies, the identification of three carbohydrate chains N-linked to surface asparagine residues, the treatment of disordered side chains and a full analysis of the solvent structure. The structure of the C. maxima laccase was refined to an R factor and R free of 18.953 and 23.835% (Table 1), respectively, applying all data to 1.9 Å, and was finally validated using the program PROCHECK (Laskowski et al., 1993). […]

Pipeline specifications

Software tools Molrep, CCP4, CNS, PROCHECK
Application Protein structure analysis
Organisms Dipturus trachyderma, Trametes versicolor
Chemicals Copper, Oxygen