Phase-diagram-guided method for growth of a large crystal of glycoside hydrolase family 45 inverting cellulase suitable for neutron structural analysis
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...
| Veröffentlicht in: | Journal of synchrotron radiation. - 1994. - 20(2013), Pt 6 vom: 07. Nov., Seite 859-63 |
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| Weitere Verfasser: | , , , , , , , , , , , |
| Format: | Online-Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
2013
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| Zugriff auf das übergeordnete Werk: | Journal of synchrotron radiation |
| Schlagworte: | Journal Article Research Support, Non-U.S. Gov't cellulase crystallization phase diagram neutron protein crystallography Glycoside Hydrolases EC 3.2.1.- Cellulase EC 3.2.1.4 |
| Zusammenfassung: | 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 |
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| Beschreibung: | Date Completed 22.05.2014 Date Revised 21.10.2021 published: Print-Electronic Citation Status MEDLINE |
| ISSN: | 1600-5775 |
| DOI: | 10.1107/S0909049513020943 |