Open AccessThe use of in situ proteolysis in the crystallization of murine CstF‐77
Author(s) -
Bai Yun,
Auperin Thierry C.,
Tong Liang
Publication year - 2007
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309107002904
Subject(s) - proteolysis , cleavage (geology) , subtilisin , protease , protein subunit , crystallization , in situ , microbiology and biotechnology , chemistry , biochemistry , biology , enzyme , paleontology , organic chemistry , fracture (geology) , gene
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.