Computational protocol: Structure of the cholera toxin secretion channel in its closed state

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Protocol publication

[…] The modeling of C12 symmetry periplasmic VcGspD rings was performed using the program SymmDock using methods previously described. A model of the pseudopilus was used based on the helical parameters from the GspK-GspI-GspJ complex, by adding additional pseudopilins to the tip. A molecular model for the export of a secreted protein by the T2SS was then obtained by aligning the helical axis of the pseudopilus with the 12-fold symmetry axis of the GspD reconstruction, and placing the structure of cholera toxin on top of the GspK-GspI-GspJ tip with the 5-fold axis of symmetry coinciding with the 12-fold and helical axes(). [...] Periplasmic VcGspD ring models were fit into the density map of VcGspD as follows. The 10 lowest energy ring models for each the N0/1, N2 and N3 domains calculated by SymmDock were initially placed into the density map of VcGspD (). The N0/N1 domain was treated as a single complex and the ring models for this domain was placed at the very bottom of the periplasmic domain of the VcGspD map, with the N-terminus pointed downward (toward the periplasm). The N2 and N3 ring models were treated separately as tethered by flexible linkers and placed in an N- to C-terminal (head-to-tail) fashion. These initial placements were optimized by automated fitting procedures in UCSF Chimera. The ring models for each domain with the best correlation to the experimental map were selected. The final N0-N3 periplasmic domain model gave a cross-correlation score of 0.65 compared the experimental map (). […]

Pipeline specifications

Software tools SymmDock, UCSF Chimera
Application Protein interaction analysis
Organisms Vibrio cholerae, Escherichia coli
Chemicals Cholera Toxin, Enterotoxins