Publications (2)16.01 Total impact
-
Article: Crystallization and preliminary X-ray crystallographic analysis of RNase HIII from Bacillus subtilis.
[show abstract] [hide abstract]
ABSTRACT: The genome of Bacillus subtilis contains three different genes encoding RNase H homologs: RNases HI, HII and HIII. RNase HIII from B. subtilis degrades RNA in RNA-DNA hybrids in an Mg(2+)-dependent manner like Escherichia coli RNase HI. However, they belong to different classes; the former belongs to the 'class II' or 'large' RNase H family, while the latter belongs to the 'class I' or 'small' RNase H family. RNase HIII of B. subtilis has been overexpressed in E. coli and crystallized at 296 K using sodium formate as a precipitant. The native X-ray diffraction data have been collected to 2.8 A resolution using synchrotron radiation. The crystals are hexagonal, belonging to the space group P6(1), with unit-cell parameters a = b = 86.89, c = 214.49 A, alpha = beta = 90.0, gamma = 120.0 degrees. A self-rotation function calculation indicated the presence of two monomers of the recombinant RNase HIII in the crystallographic asymmetric unit, giving a V(M) of 3.43 A(3) Da(-1) and a solvent content of 64.2%.Acta Crystallographica Section D Biological Crystallography 04/2001; 57(Pt 3):438-40. · 12.62 Impact Factor -
Article: Crystallization, molecular replacement solution, and refinement of tetrameric beta-amylase from sweet potato.
[show abstract] [hide abstract]
ABSTRACT: Sweet potato beta-amylase is a tetramer of identical subunits, which are arranged to exhibit 222 molecular symmetry. Its subunit consists of 498 amino acid residues (Mr 55,880). It has been crystallized at room temperature using polyethylene glycol 1500 as precipitant. The crystals, growing to dimensions of 0.4 mm x 0.4 mm x 1.0 mm within 2 weeks, belong to the tetragonal space group P4(2)2(1)2 with unit cell dimensions of a = b = 129.63 A and c = 68.42 A. The asymmetric unit contains 1 subunit of beta-amylase, with a crystal volume per protein mass (VM) of 2.57 A3/Da and a solvent content of 52% by volume. The three-dimensional structure of the tetrameric beta-amylase from sweet potato has been determined by molecular replacement methods using the monomeric structure of soybean enzyme as the starting model. The refined subunit model contains 3,863 nonhydrogen protein atoms (488 amino acid residues) and 319 water oxygen atoms. The current R-value is 20.3% for data in the resolution range of 8-2.3 A (with 2 sigma cut-off) with good stereochemistry. The subunit structure of sweet potato beta-amylase (crystallized in the absence of alpha-cyclodextrin) is very similar to that of soybean beta-amylase (complexed with alpha-cyclodextrin). The root-mean-square (RMS) difference for 487 equivalent C alpha atoms of the two beta-amylases is 0.96 A. Each subunit of sweet potato beta-amylase is composed of a large (alpha/beta)8 core domain, a small one made up of three long loops [L3 (residues 91-150), L4 (residues 183-258), and L5 (residues 300-327)], and a long C-terminal loop formed by residues 445-493. Conserved Glu 187, believed to play an important role in catalysis, is located at the cleft between the (alpha/beta)8 barrel core and a small domain made up of three long loops (L3, L4, and L5). Conserved Cys 96, important in the inactivation of enzyme activity by sulfhydryl reagents, is located at the entrance of the (alpha/beta)8 barrel.Proteins Structure Function and Bioinformatics 03/1995; 21(2):105-17. · 3.39 Impact Factor
