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] |
| Copyright of Journal of Synchrotron Radiation is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.) | |
| Database: | Engineering Source |
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| Header | DbId: egs DbLabel: Engineering Source An: 90674216 AccessLevel: 6 PubType: Academic Journal PubTypeId: academicJournal PreciseRelevancyScore: 0 |
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| Items | – Name: Title Label: Title Group: Ti Data: Phase-diagram-guided method for growth of a large crystal of glycoside hydrolase family 45 inverting cellulase suitable for neutron structural analysis. – Name: Author Label: Authors Group: Au Data: <searchLink fieldCode="AR" term="%22Nakamura%2C+Akihiko%22">Nakamura, Akihiko</searchLink><br /><searchLink fieldCode="AR" term="%22Ishida%2C+Takuya%22">Ishida, Takuya</searchLink><br /><searchLink fieldCode="AR" term="%22Fushinobu%2C+Shinya%22">Fushinobu, Shinya</searchLink><br /><searchLink fieldCode="AR" term="%22Kusaka%2C+Katsuhiro%22">Kusaka, Katsuhiro</searchLink><br /><searchLink fieldCode="AR" term="%22Tanaka%2C+Ichiro%22">Tanaka, Ichiro</searchLink><br /><searchLink fieldCode="AR" term="%22Inaka%2C+Koji%22">Inaka, Koji</searchLink><br /><searchLink fieldCode="AR" term="%22Higuchi%2C+Yoshiki%22">Higuchi, Yoshiki</searchLink><br /><searchLink fieldCode="AR" term="%22Masaki%2C+Mika%22">Masaki, Mika</searchLink><br /><searchLink fieldCode="AR" term="%22Ohta%2C+Kazunori%22">Ohta, Kazunori</searchLink><br /><searchLink fieldCode="AR" term="%22Kaneko%2C+Satoshi%22">Kaneko, Satoshi</searchLink><br /><searchLink fieldCode="AR" term="%22Niimura%2C+Nobuo%22">Niimura, Nobuo</searchLink><br /><searchLink fieldCode="AR" term="%22Igarashi%2C+Kiyohiko%22">Igarashi, Kiyohiko</searchLink><br /><searchLink fieldCode="AR" term="%22Samajima%2C+Masahiro%22">Samajima, Masahiro</searchLink> – Name: TitleSource Label: Source Group: Src Data: <searchLink fieldCode="JN" term="%22Journal+of+Synchrotron+Radiation%22">Journal of Synchrotron Radiation</searchLink>. Nov2013, Vol. 20 Issue 6, p859-863. 5p. – Name: Subject Label: Subjects Group: Su Data: <searchLink fieldCode="DE" term="%22Neutron+diffraction+crystallography%22">Neutron diffraction crystallography</searchLink><br /><searchLink fieldCode="DE" term="%22X-ray+crystallography%22">X-ray crystallography</searchLink><br /><searchLink fieldCode="DE" term="%22Glycosidases%22">Glycosidases</searchLink><br /><searchLink fieldCode="DE" term="%22Crystallization%22">Crystallization</searchLink><br /><searchLink fieldCode="DE" term="%22Nucleation%22">Nucleation</searchLink><br /><searchLink fieldCode="DE" term="%22Precipitation+%28Chemistry%29%22">Precipitation (Chemistry)</searchLink> – Name: Abstract Label: Abstract Group: Ab Data: 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] – Name: AbstractSuppliedCopyright Label: Group: Ab Data: <i>Copyright of Journal of Synchrotron Radiation is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.) |
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| RecordInfo | BibRecord: BibEntity: Identifiers: – Type: doi Value: 10.1107/S0909049513020943 Languages: – Code: eng Text: English PhysicalDescription: Pagination: PageCount: 5 StartPage: 859 Subjects: – SubjectFull: Neutron diffraction crystallography Type: general – SubjectFull: X-ray crystallography Type: general – SubjectFull: Glycosidases Type: general – SubjectFull: Crystallization Type: general – SubjectFull: Nucleation Type: general – SubjectFull: Precipitation (Chemistry) Type: general Titles: – TitleFull: Phase-diagram-guided method for growth of a large crystal of glycoside hydrolase family 45 inverting cellulase suitable for neutron structural analysis. Type: main BibRelationships: HasContributorRelationships: – PersonEntity: Name: NameFull: Nakamura, Akihiko – PersonEntity: Name: NameFull: Ishida, Takuya – PersonEntity: Name: NameFull: Fushinobu, Shinya – PersonEntity: Name: NameFull: Kusaka, Katsuhiro – PersonEntity: Name: NameFull: Tanaka, Ichiro – PersonEntity: Name: NameFull: Inaka, Koji – PersonEntity: Name: NameFull: Higuchi, Yoshiki – PersonEntity: Name: NameFull: Masaki, Mika – PersonEntity: Name: NameFull: Ohta, Kazunori – PersonEntity: Name: NameFull: Kaneko, Satoshi – PersonEntity: Name: NameFull: Niimura, Nobuo – PersonEntity: Name: NameFull: Igarashi, Kiyohiko – PersonEntity: Name: NameFull: Samajima, Masahiro IsPartOfRelationships: – BibEntity: Dates: – D: 01 M: 11 Text: Nov2013 Type: published Y: 2013 Identifiers: – Type: issn-print Value: 09090495 Numbering: – Type: volume Value: 20 – Type: issue Value: 6 Titles: – TitleFull: Journal of Synchrotron Radiation Type: main |
| ResultId | 1 |
