Crystallization of Synthetic Escherichia coli‐Type Lipid A

Abstract

Synthetic Escherichia coli-type lipid A formed hexagonal plate crystals when it was precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl2 and incubated in 70% ethanol containing 250 mM MgCl2 at 4 C for 10 days. Analyses of crystals by electron diffraction and synchrotron X-ray diffraction showed that crystals consist of hexagonal lattices with the lattice constant (a side of the lozenge as a unit cell on the basal plane) of 4.62 angstrom and the longitudinal axis (perpendicular to the basal plane) of 49.3 +/- 1.3 angstrom. Results suggest that the previous finding that various kinds of R-form lipopolysaccharides crystallized but free lipid A isolated by acid hydrolysis from Re lipopolysaccharide did not crystallize under the same experimental conditions (Kato et al, J. Bacteriol., 172:1516-1528, 1990) is due to structural changes of lipid A occurring during the procedure of isolation of free lipid A.

Crystallization of Lipopolysaccharide from a Salmonella typhimurium Semi‐Rough (SR) Mutant

“…Electron microscopy. For observation of morphology of crystals and analysis of crystals by electron diffraction, an electron microscope (JEM-2000FX, JEOL, Ltd., Akishima, Japan), operating at 200 kV and equipped with a cryo-transfer holder which is cooled with liquid nitrogen, was used as in the previous study (4,5). On the preparation of specimens to be tested, a drop of crystals was placed on a microgrid coated with carbon and then gold, and the liquid was removed by filter paper from the reverse side of the grid.…”

“…For comparison, crystals of synthetic E. colitype lipid A (1,2) was also subjected to X-ray diffraction analysis under the same experimental conditions except that the wavelength of the X-ray was set to 1.00A. Detailed results of the X-ray analysis of crystals of the synthetic lipid A have already been reported (5).…”

“…and E. coli and synthetic E. co/i-type lipid A were characteristic of polycrystals and consisted of a series of inner ring-shaped reflections and an outermost ring-shaped reflection (5,8). The inner ring-shaped reflections proved to be due to a periodicity along the longitudinal axis (c axis) and the outermost ring corresponded to a periodicity of 4.0 A (a axis of 4.62 A) (5,8). X-ray diffraction patterns in the low-angle region from the crystals of SR-form LPS and synthetic E. co/i-type lipid A are shown in Fig.…”

“…Later, it was found that synthetic hexaacyl lipid A (E. coli-type lipid A), which is considered to be the real minimal endotoxic material of LPS, not only from E. coli but also from Salmonella spp. (10,15), could crystallize under the same experimental conditions (5). Therefore, the failure to crystallize free lipid A isolated by acid hydrolysis is due to structural changes in lipid A occurring as the result of chemical treatments used for the isolation process.…”

Crystallization of an R‐Form Lipopolysaccharide from Klebsiella pneumoniae

“…Furthermore, we recently revealed that synthetic E. colitype lipid A forms hexagonal plate crystals which consist of hexagonal lattices with the same a-axis value as crystals of R-form LPSs from Salmonella spp. and E. coli (6).…”