Extended Data Fig. 7: Cryo-EM structures of substrate-free NSF in the hydrolyzing condition.

a-d, Cryo-EM maps (sharpened cryoSPARC) in three orientations (top to bottom rows) of the four classes of NSF in the hydrolyzing condition. The NSF heptamer (class 1) is designated as class 1. Classes 2-4 showed hexameric NSF in similar orientations with varying resolution for the substrate, so the best one, class 3 (initial) (c), was chosen for 3DVA analysis in CryoSPARC. e, The main principal component of the 3DVA analysis was used to select a subset of particles clearly showing an NSF N-terminal domain that is engaged with the D2 ring and then re-refined against this subset of particles to yield the final map for class 3. f-g, insets showing that an N-domain acts as a bound substrate in class 3 (final).