MOLECULAR REPLACEMENT AT ITS LIMITS? THE STRUCTURE DETERMINATION OF T.cruzi g-GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE, 12 MONOMERS IN THE ASYMMETRIC UNIT AND 41% DATA COMPLETENESS AT 3.5 Å RESOLUTION. 1Guimarães, B.G., 1,2Souza, D.H.F, 1Jesus,W.D.P. and 1Oliva, G., 1. Instituto de Física de São Carlos, USP, Cx.P.369, 13560, São Carlos, SP, Brazil; 2. Instituto de Química de São Carlos, USP.
Recent developments in the software tools available for the Molecular Replacement Technique (AMoRe, Navaza, 1994, Acta Cryst. A50, 157-163) have dramatically improved the efficiency and speed of the calculations, making it possible to explore a very large number of potential solutions, even in the limits of partial search models and incompleteness of diffraction data. In the search for crystallization conditions of the trypanosomal enzyme gGAPDH, a crystal form was obtained belonging to space group I4, with very large cell parameters a=b=307.93 Å, c=119.82 Å and diffracting to low resolution (3.5 Å). The crystal has 12 monomers per a.u., Vm= 3.035 Å3.dalton-1 and 59.5% solvent. High sensitivity to radiation and lack of isomorphism between different native crystals, results in severe limitations in data collection. Data was measured at station BW6 at HASYLAB-DESY. From the seven crystals used, only two could be merged after integration. Merging of the 35139 measured reflections resulted in 29097 independent reflections (41% completeness), Rmerge= 12.1%. The structure was solved with AMoRe, using the tetramer of T.cruzi gGAPDH as determined from crystal form I (space group P1). 250 peaks from the rotation function had to be included to find the correct solution, highlighting the importance of assessing a large number of possible solutions. Details of the constrained refinement will also be presented.
Acknowledgements: Dr.J.Navaza, WHO (TDR grant 940854), PADCT/SBIO , CNPq, FAPESP and FINEP.