The use of in situ proteolysis in the crystallization of murine CstF-77.
The cleavage-stimulation factor (CstF) is required for the cleavage of the 3'-end of messenger RNA precursors in eukaryotes. During structure determination of the 77 kDa subunit of the murine CstF complex (CstF-77), it was serendipitously discovered that a solution infected by a fungus was crucial for the crystallization of this protein. CstF-77 was partially proteolyzed during crystallization; this was very likely to have been catalyzed by a protease secreted by the fungus. It was found that the fungal protease can be replaced by subtilisin and this in situ proteolysis protocol produced crystals of sufficient size for structural studies. After an extensive search, it was found that 55% glucose can be used as a cryoprotectant while maintaining the diffraction quality of the crystals; most other commonly used cryoprotectants were detrimental to the diffraction quality.
Bai
Yun
Y
Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Auperin
Thierry C
TC
Tong
Liang
L
D000818:Animals; D039241:Cleavage Stimulation Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003460:Crystallization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D039241:Cleavage Stimulation Factor; D021122:Protein Subunits; D010447:Peptide Hydrolases