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 ...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.展开更多
To achieve de novo protein structure determination of challenging cases, multi-wavelength anomalous diffraction(MAD) and multiple isomorphous replacement(MIR) phasing can be powerful tools to obtain low-resolution ini...To achieve de novo protein structure determination of challenging cases, multi-wavelength anomalous diffraction(MAD) and multiple isomorphous replacement(MIR) phasing can be powerful tools to obtain low-resolution initial phases from heavy-atom derivative datasets, then phase extension is needed against high-resolution data to obtain accurate structures.In this context, we propose a direct-methods procedure here that could improve the initial low-resolution MAD/MIR phase quality.And accordingly, an automated process for extending initial phases to high resolution is also described.These two procedures are both implanted in the newly released IPCAS pipeline.Three cases are used to perform the test, including one set of 4.17 ? MAD data from a membrane protein and two sets of MAD/MIR data with derivatives truncated down to 6.80 ? and 6.90 ?, respectively.All the results have shown that the initial phases generated from the direct-methods procedure are better than that from the conventional MAD/MIR methods.The automated phase extensions for the latter two cases starting from 6.80 ? to 3.00 ? and 6.90 ? to 2.80 ? are proved to be successful, leading to complete models.This may provide convenient and reliable tools for phase improvement and phase extension in difficult low-resolution tasks.展开更多
基金supported by the Key Project Foundation of the Chinese Academy of Sciences(Project No.KJ951-A1-601)the National Natural Science Foundation of China(Grant No.B705975)the National Key Research Development Project of China(Project No.G1999075601).
文摘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.
基金Project supported by the National Basic Research Program of China(Grant No.2011CB911100)of the Ministry of Science and Technology of China
文摘To achieve de novo protein structure determination of challenging cases, multi-wavelength anomalous diffraction(MAD) and multiple isomorphous replacement(MIR) phasing can be powerful tools to obtain low-resolution initial phases from heavy-atom derivative datasets, then phase extension is needed against high-resolution data to obtain accurate structures.In this context, we propose a direct-methods procedure here that could improve the initial low-resolution MAD/MIR phase quality.And accordingly, an automated process for extending initial phases to high resolution is also described.These two procedures are both implanted in the newly released IPCAS pipeline.Three cases are used to perform the test, including one set of 4.17 ? MAD data from a membrane protein and two sets of MAD/MIR data with derivatives truncated down to 6.80 ? and 6.90 ?, respectively.All the results have shown that the initial phases generated from the direct-methods procedure are better than that from the conventional MAD/MIR methods.The automated phase extensions for the latter two cases starting from 6.80 ? to 3.00 ? and 6.90 ? to 2.80 ? are proved to be successful, leading to complete models.This may provide convenient and reliable tools for phase improvement and phase extension in difficult low-resolution tasks.
