title: Phase-diagram-guided method for growth of a large crystal of glycoside hydrolase family 45 inverting cellulase suitable for neutron structural analysis.
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 mM tris-HCl pH 8.0) with a 1,000 µl reservoir (61% 3-methyl-1,5,-pentanediol in 50 mM 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(3) volume (3 mm × 2 mm × 1 mm), which was suitable for neutron diffraction.
authors.affiliation: Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Nakamura
authors.forename: Takuya
authors.initials: T
authors.lastname: Ishida
authors.forename: Shinya
authors.initials: S
authors.lastname: Fushinobu
authors.forename: Katsuhiro
authors.initials: K
authors.lastname: Kusaka
authors.forename: Ichiro
authors.initials: I
authors.lastname: Tanaka
authors.forename: Koji
authors.initials: K
authors.lastname: Inaka
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Higuchi
authors.forename: Mika
authors.initials: M
authors.lastname: Masaki
authors.forename: Kazunori
authors.initials: K
authors.lastname: Ohta
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kaneko
authors.forename: Nobuo
authors.initials: N
authors.lastname: Niimura
authors.forename: Kiyohiko
authors.initials: K
authors.lastname: Igarashi
authors.forename: Masahiro
authors.initials: M
authors.lastname: Samajima
mesh_terms: D002480:Cellulase / Q000737:chemistry*; D018360:Crystallography, X-Ray; D006026:Glycoside Hydrolases / Q000737:chemistry*; D009502:Neutrons
chemical_list: D006026:Glycoside Hydrolases; D002480:Cellulase
keywords: cellulase; crystallization phase diagram; neutron protein crystallography