ClpAP is a barrel-like complex consisting of hexameric rings of the ClpA ATPase stacked on the double heptameric ring of ClpP peptidase. ClpA has two AAA+ domains (D1 and D2) and a 153-residue N-domain. Substrate proteins bind to the distal surface of ClpA and are unfolded and translocated axially into ClpP. To gain insight into the functional architecture of ClpA in the ATPgammaS state, we have determined its structure at 12 A resolution by cryo-electron microscopy. The resulting model has two tiers, corresponding to rings of D1 and D2 domains: oddly, there is no sign of the N-domains in the density map. However, they were detected as faint diffuse density distal to the D1 tier in a difference image between wild-type ClpAP and a mutant lacking the N-domain. This region is also accentuated in a variance map of ClpAP and in a difference imaging experiment with ClpAP complexed with ClpS, a 12 kDa protein that binds to the N-domain. These observations demonstrate that the N-domains are highly mobile. From molecular modeling, we identify their median position and estimate that they undergo fluctuations of at least 30 Angstrom. We discuss the implications of these observations for the role of N-domains in substrate binding: either they effect an initial transient binding, relaying substrate to a second site on the D1 tier where unfolding ensues, or they may serve as an entropic brush to clear the latter site of non-specifically bound ligands or substrates bound in non-productive complexes. Published by Elsevier Inc.