Structural basis for the recognition of K48-linked Ub chain by proteasomal receptor Rpn13

a With fluorophores conjugated at 76？C site of the proximal Ub and 0？C of the distal Ub (cf. Supplementary Fig. S1a), the smFRET profile can be fitted to three overlapping FRET species. Unless otherwise noted, this pair of conjugation sites are used for all smFRET studies of K48-diUb. The high-FRET species (colored red) corresponds to a preexisting compact state. b–d The compact state can be selectively enriched by full-length Rpn13, and the population increase can be fitted to a binding isotherm. e–g The compact state can be selectively enriched by Rpn13NTD, and the population increase can be fitted to be a binding isotherm. h With the fluorophores conjugated at N25C/N25C sites, Rpn13NTD can selectively enrich the preexisting compact state of the distal diUb of K48-tetraUb (cf. Supplementary Fig. S5), affording a binding affinity. The populations of the smFRET species are averaged over three independent measurements, with the errors indicating 1？SD; the KD values are reported as best fit？±？fitting error