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Cysteine cross-linking of JetA/JetC interface residues. Top: imaging of TMR-labeled JetA-HaloTag after BMOE cross-linking of JetABC in the indicated conditions. (A–C) JetA-JetC cross-linked product; (A) JetA. Of note, we also readily detected JetA-JetA cross-linked product (indicated by asterisk), stemming from JetA(E229C) cross-linking, possibly between dimers of pentamers. Alternatively, the cross-linking may result from an unknown (intermediary) conformation(s) of the complex. Bottom: position of the engineered cysteine residues shown as balls in green colors in the models for the resting (PDB:8BFN) and cleavage-competent states. The yellow dashed lines indicate the Cα-Cα distances between selected cysteine pairs. See alsoFigures S4–S7andTable 1.

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