Extended Data Fig. 5: Assembly and crystallization of a USP54-diUb(K63)-PA complex.
From: Discovery and mechanism of K63-linkage-directed deubiquitinase activity in USP53
a. Fluorescence raw data of the protein stability assessment. Corresponding melting temperatures are shown in Fig. 4c. b. Assembly and purification strategy to obtain a pure USP54~diUb(K63)-PA complex for crystallization. Of note, cleavage of the native isopeptide bond in the probe was suppressed by protein labeling at low temperature and with an excess of probe over enzyme. c. Deconvoluted intact protein mass spectra of the K63-linked diUb-PA probe, USP54, and the USP54~diUb(K63)-PA complex. Calculated and observed molecular weights are given in Dalton. d. Asymmetric unit (a.s.u.) of the crystal structure of USP54~diUb-PA. All four copies of USP54~diUb-PA are shown in cartoon representation. e. Asymmetric unit (a.s.u.) of the crystal structure of USP54~diUb-PA as in panel d, all four copies are shown in stick representation overlayed with 2FO-FC electron density contoured at 1 σ. f. Chains ABCDEF and chains GHIJKL, each consisting of 2x USP54~diUb-PA, are shown individually and as an overlay in cartoon representation.
