Delta endotoxins are the entomocidal agent produced by Bacillus thuringiensis ssp. Kurstaki. These toxins are produced as protoxins (135 kDa) by the bacteria during sporulation and recognized by their bipyrimidal crystalline form in the sporangium. When ingested by larval Lepidoptera the crystal dissolves and is proteolytically digested to the active toxin form (60-65 kDa). This activated toxin is capable of opening K+ channels in the epithelium of the insect midgut. Ion leakage ultimately results in the complete disruption of the midgut and death of the insect. More recently, a new active form of the toxin, CryIIIB2, has been determined (64 kDa). The bacterial endotoxin CryIIIB2 is a 652 residue protein which crystallizes in the orthorhombic space group C2221 with unit cell dimensions a = 122.44, b = 131.81, and c = 105.37& and contain one molecule in the asymmetric unit. Synchrontron radiation was used to collect 2.2& resolution data at the CHESS facility at Cornell University in December, 1997 by Vivian Cody and Nikolai Galitsky for the complex of EC11095 with D-galactosamine. Although these data were did not diffract uniformly to high resolution, Walt Pangborn was able to scale data with at least 50% completion for all shells to 2.2& resolution. The structure of the native CryIIIB2 was used as a search model for the rotation and translation function in the program XPLOR which revealed there was no significant change in the molecular orientation of the molecule compared to the room temperature model. The major consequence of low temperature data collection is the contraction of the unit cell along the a and b lattice directions. Interpretation of the difference electron density maps made from the 3.0& refinement model of the Arg-348 mutant of CryIIIB2 revealed a large density profile that clearly was not a cluster of solvent and that could be fit to the sugar. These data reveal that the galactosamine is positioned at the intersection of the three domains of CryIIIB2. Refinement of these data to 2.2& resolution suggest other potential sugar binding sites are possible.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001646-19
Application #
6491104
Study Section
Project Start
2001-08-15
Project End
2002-08-14
Budget Start
Budget End
Support Year
19
Fiscal Year
2001
Total Cost
$142,703
Indirect Cost
Name
Cornell University
Department
Type
DUNS #
City
Ithaca
State
NY
Country
United States
Zip Code
14850
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