Supplementary Figure 1: Purification, characterization and cryo-EM analysis of the human 26S proteasome.

(a) A schematic diagram of the affinity purification protocol for endogenous human proteasome from a modified HEK293T cell line. (b) The purified human proteasome is reasonably homogeneous on native PAGE and exhibits proteolytic activity. The purified human proteasome appeared mainly as a dominant band of a 2:1 complex between the RP and the CP on native PAGE (lane 1). Incubation of the native PAGE gel with the fluorogenic substrate Suc-LLVY-AMC revealed substrate degradation as detected by fluorescence emission of the free AMC moiety (lane 2). (c) Identification of the Rpn and Rpt subunits on SDS-PAGE. The purified human proteasome was applied to SDS-PAGE followed by Coomassie staining (lane 1). The molecular weight markers are shown in lane 2. (d) Results of mass spectrometric (MS) identification of the human proteasomal subunits. All subunits were identified by MS. (e) A representative cryo-EM micrograph of the human proteasome. The proteasome particles are clearly seen in the original micrograph. Scale bar, 50 nm. (f) Representative 2D class averages of the human proteasome. Due to its elongated shape, the proteasome displays a clear preference of orientation on the carbon-coated grid.