Phase‐diagram‐guided method for growth of a large crystal of glycoside hydrolase family 45 inverting cellulase suitable for neutron structural analysis. (25th September 2013)

• Record Type: Journal Article
• 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
• Is Part Of: Journal of synchrotron radiation. Volume 20:Part 6(2013)
• Page Start: 859
• Page End: 863
• Publication Date: 2013-09-25
• Subjects: Synchrotron radiation -- Periodicals; Free electron lasers -- Periodicals; 539.73505
• DOI: 10.1107/S0909049513020943
• Languages: English
• ISSNs: 0909-0495

Abstract

<abstract abstract-type="main" xml:lang="en"> <title> <x xml:space="preserve">Abstract</x> </title> <p>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 <italic>Pc</italic>Cel45A 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<sup>−1</sup> protein with 30.5% 3‐methyl‐1, 5‐pentanediol in 50 m<italic>M</italic> tris‐HCl pH 8.0) with a 1000 µl reservoir (61% 3‐methyl‐1, 5, ‐pentanediol in 50 m<italic>M</italic> 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 mm<sup>3</sup> volume (3 mm × 2 mm × 1 mm), which was suitable for neutron diffraction.</p> </abstract>