Fig. 3: Domain assignments in negative stain 2D class averages of Rea1_ΔAAA2H2α.

a CryoEM map (left panels) and cartoon representation (right panels) of Rea1_ΔAAA2H2α in the presence of ATPγS. The red arrow heads highlight the AAA+ ring docked MIDAS domain. b Schematic cartoon representation of the structure in a. The α-helical extension of AAA2 normally occupying the centre of the pore (compare Fig. 1b) has been deleted, which allows the MIDAS domain to dock onto the AAA+ ring. The D/E rich region connecting the MIDAS domain to the linker top is flexible and not visible in the structure. Adapted from Sosnowski et al.¹⁷. Elife 7, https://doi.org/10.7554/eLife.39163 (2018) under a CC BY license: https://creativecommons.org/licenses/by/4.0/. c Upper panels: A 2D projection of the cryoEM map in A. low pass filtered to 25 Å shows a good match for the AAA+ ring region in the state 1 negative stain 2D class average. The projection allows the assignment of the NTD, AAA1–AAA6, the linker stem and the MIDAS domain. In contrast to the AAA+ ring region, the linker top and middle domains adopt a different conformation from the one seen in state 1. Lower panels: With a different 2D projection of the low pass filtered cryoEM map, a good match for the linker region in the state 1 negative stain 2D class average can be produced, allowing the assignment of the linker top1, top2 and top3 domains as well as the linker middle domain. The AAA+ ring does not match up with the AAA+ ring in the state 1 negative stain 2D class average. This mismatch indicates that -compared to the cryoEM structure in A. - the linker top and middle domains in state 1 have moved with respect to the AAA+ ring. d The assignment of the MIDAS domain in state 1 of Rea1_ΔAAA2H2α (red arrow head, left panel) is further supported by comparisons with state 1 of Rea1_wt, where the MIDAS domain is absent from the AAA+ ring (middle panel, compare also Supplementary Fig. 11) as well as analysis of the Rea1_{ΔAAA2H2α+ΔMIDAS} double mutant (right panel).