Multi-isomorphous replacement phasing of the earthworm fibrinolytic enzyme component A from Eisenia fetida

【Abstract】 Earthworm fibrinolytic enzyme component A (EFEa) from Eisenia fetida, a protein func-tioning not only as a direct fibrinolytic enzyme, but also as a plasminogen activator, has been crystallized in P212121 space group with 3 protein molecules per asymmetric unit. Four heavy atom derivatives were prepared using a mother liquor containing 1.4 mol·L-1 Li2SO4 and 0.1 mol·L-1 MOPS buffer (pH7.2) and used to solve the protein抯 diffraction phase. The heavy atom binding sites in the derivative crystals were determined using difference Patterson and difference Fourier methods and were refined in combination to yield the initial protein抯 structure phase at 0.25 nm resolution. The non-crystallographic symmetry relationship of the three independent protein mole-cules in the asymmetric unit was determined using the correlative heavy atom sites and used for the averaging of the initial electron density. As a result, the electron density was significantly im-proved, providing a solid foundation for subsequent structure determination.更多还原