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Title: The Use of in situ Proteolysis in the Crystallization of Murine CstF-77

Abstract

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.

Authors:
; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
930333
Report Number(s):
BNL-81044-2008-JA
Journal ID: ISSN 1744-3091; TRN: US200904%%627
DOE Contract Number:  
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica Section F: Structural Biology and Crystallization Communications; Journal Volume: 63
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; CLEAVAGE; CRYSTALS; CRYSTAL STRUCTURE; CRYSTALLIZATION; DIFFRACTION; GLUCOSE; MESSENGER-RNA; PROTEINS; PROTEOLYSIS; RODENTS; national synchrotron light source

Citation Formats

Bai,Y., Auperin, T., and Tong, L. The Use of in situ Proteolysis in the Crystallization of Murine CstF-77. United States: N. p., 2007. Web. doi:10.1107/S1744309107002904.
Bai,Y., Auperin, T., & Tong, L. The Use of in situ Proteolysis in the Crystallization of Murine CstF-77. United States. doi:10.1107/S1744309107002904.
Bai,Y., Auperin, T., and Tong, L. Mon . "The Use of in situ Proteolysis in the Crystallization of Murine CstF-77". United States. doi:10.1107/S1744309107002904.
@article{osti_930333,
title = {The Use of in situ Proteolysis in the Crystallization of Murine CstF-77},
author = {Bai,Y. and Auperin, T. and Tong, L.},
abstractNote = {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.},
doi = {10.1107/S1744309107002904},
journal = {Acta Crystallographica Section F: Structural Biology and Crystallization Communications},
number = ,
volume = 63,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}