Mini-Symposium - Abstracts
6 CRYSTAL STRUCTURE OF A PHOSPHOLIPASE A2 FROM Bothrops moojeni
M.C. NONATO, R.C. GARRATT, Y.P. MASCARENHAS, G. OLIVA
Laboratory of Crystallography, Institute of Physics of São Carlos, University of São Paulo, Brazil
Phospholipase A2 from Bothrops moojeni (BM-PLA2) was purified (Reichel et al., 1989, Mem. Inst. Butantan 51(4), 225-237) and crystallized by vapor diffusion techniques in hanging drops using 1ml of reservoir solution containing from 34% to 44% of a 10:90 sodium phosphate monobasic and potassium phosphate monobasic solution. BM-PLA2 crystals belong to the monoclinic system, space group C2 with cell dimensions a=120.4, b=59.6, c=96.6Å and ß=125º, four molecules in the asymmetric unit for an estimated solvent content of 60%. The crystals showed to be very sensitive to X-ray exposure. A preliminary data collection from one crystal of approximately size of 0.4 x 0.2 x 0.8 mm provided a data set with 6402 independent reflections, 50.7% completeness, Rmerge =15.2% for a multiplicity of 1.33 to 2.9Å resolution.
The structure was solved by molecular replacement techniques using the AMoRe package. The dimer of Crotalus atrox was used as a model providing a distinctive peak with a correlation factor of 50.0% with data to 3.5Å resolution. The initial refinement, which includes rigid body refinement, density averaging, manual manipulations were residues without clear density were mutated to alanine and those showing clear density were modeled into the electron density and simulated annealing provided a preliminary model with Rfactor of 28.3% and Rfree of 33.4%. The analysis of an averaged map showed that all the disulfide bridges present in the Crolatus atrox structure are conserved .The electron density for the residues in the active site is very well defined. The presence of the calcium ion is still in discussion. New cycles of refinement will be made followed by new purification and crystallization experiments.
CORRESPONDENCE TO:
Dra. Maria Cristina Nonato - Departamento de Física e Informática, Instituto de Física de São Carlos, USP, CEP 13560-970, Caixa Postal 369, São Carlos, SP, Brasil. email: cristy@ifqsc.sc.usp.br
Publication Dates
-
Publication in this collection
08 Jan 1999 -
Date of issue
1997
