Phase-diagram-guided method for growth of a large crystal of glycoside hydrolase family 45 inverting cellulase suitable for neutron structural analysis.
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| Title: | Phase-diagram-guided method for growth of a large crystal of glycoside hydrolase family 45 inverting cellulase suitable for neutron structural analysis. |
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| Authors: | Nakamura, Akihiko, Ishida, Takuya, Fushinobu, Shinya, Kusaka, Katsuhiro, Tanaka, Ichiro, Inaka, Koji, Higuchi, Yoshiki, Masaki, Mika, Ohta, Kazunori, Kaneko, Satoshi, Niimura, Nobuo, Igarashi, Kiyohiko, Samajima, Masahiro |
| Source: | Journal of Synchrotron Radiation. Nov2013, Vol. 20 Issue 6, p859-863. 5p. |
| Subjects: | Neutron diffraction crystallography, X-ray crystallography, Glycosidases, Crystallization, Nucleation, Precipitation (Chemistry) |
| Abstract: | Neutron protein crystallography (NPC) is a powerful tool for determining the hydrogen position and water orientation in proteins, but a much larger protein crystal is needed for NPC than for X-ray crystallography, and thus crystal preparation is a bottleneck. To obtain large protein crystals, it is necessary to know the properties of the target protein in the crystallization solution. Here, a crystal preparation method of fungal cellulase PcCel45A is reported, guided by the phase diagram. Nucleation and precipitation conditions were determined by sitting-drop vapor diffusion. Saturation and unsaturation conditions were evaluated by monitoring crystal dissolution, and a crystallization phase diagram was obtained. To obtain a large crystal, crystallization solution was prepared on a sitting bridge (diameter = 5 mm). Initial crystallization conditions were 40 µl of crystallization solution (40 mg ml−1 protein with 30.5% 3-methyl-1,5-pentanediol in 50 m M tris-HCl pH 8.0) with a 1000 µl reservoir (61% 3-methyl-1,5,-pentanediol in 50 m M tris-HCl pH 8.0) at 293 K. After the first crystal appeared, the concentration of precipitant in the reservoir solution was reduced to 60% to prevent formation of further crystals. Finally, we obtained a crystal of 6 mm3 volume (3 mm × 2 mm × 1 mm), which was suitable for neutron diffraction. [ABSTRACT FROM AUTHOR] |
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| Database: | Engineering Source |
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