Extended Data Fig. 7: Further cryo-EM data processing of resting-state (RS) proteasomes with bound TXNL1 in continuation from Extended Data Fig. 6.
From: Structural landscape of the degrading 26S proteasome reveals conformation-specific binding of TXNL1
a) ‘Slight left’ and ‘Slight right’ particles were processed separately. Particle stacks were aligned (local refinement in CryoSparc) with a local mask surrounding the PITH domain, Rpn2, Rpn10, Rpn11, and the N-ring of the Rpt hexamer. CryoSparc 3D variability analysis of aligned particles with the local mask, followed by 3D cluster analysis, allowed grouping of particles into two distinct conformations, forward and backward with respect to the PITH domain position. A third group of particles showed the PITH domain in variable positions and could not be assigned to a particular state, suggesting that the PITH domain has continuous motion between forward and backward orientations. b) Local refinement with a mask surrounding the whole RP resulted in 4 distinct conformations defined by a slight shift in the RP and the two orientations of the PITH domain: RS.1 TXNL1 Forward (RS.1 state 1), RS.1 TXNL1 Backward (RS.1 state 2), RS.2 TXNL1 Forward (RS.2 state 1), and RS.2 TXNL1 Backward (RS.2 state 2). Each structure was resolved to high resolution, as demonstrated by the maps colored according to estimations of local resolutions, calculated with CryoSparc using a 0.143 FSC cutoff. c) Alignments of the atomic models for RS.1 and RS.2, states 1 and 2, highlight the shift in the RP, in addition to variations in Rpn1’s position. Right: Particle numbers for each conformational state that were used to calculate the percentages of proteasomes with TXNL1 in the forward, backward, or mixed orientations shown in Fig. 2b.
