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Left: schematic of Cdc48-UN with a ubiquitin moiety (yellow circle) proximal to the initiator ubiquitin (red line) interacting with the UT3 domain (purple) of the Ufd1 cofactor subunit. Placing a Cy3 donor dye on the N terminus of this proximal ubiquitin and a Cy5 acceptor on UT3 (R64C) allows monitoring this interaction through FRET. Right: AlphaFold2/ColabFold model for the complex between a K48-linked ubiquitin and Ufd1’s UT3 domain. I44 and K48 of ubiquitin, as well as the binding-relevant L55 of UT3 are depicted as blue, red, and orange sticks, respectively. For PAE plot seeFigure S3.

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