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Reca Assembly, One Molecule at a Time

is reversible; if substrate were removed, would the majority of particles return to the standard conformation? The expanded structure is highly deformed from the standard state, but the relatively modest resolution of the reconstructions (12–16 Å), due to unresolved heterogeneity in the dataset, means that interpretation of what exactly happens to the GroEL domains is unclear, although the apical and intermediate domains appear to expand outwards, and equatorial domains slide inwards. Until now, conformational changes due to nucleotide and GroES binding were characterized as rigid-body movements (albeit large ones) of the intermediate and apical domains. This expanded state shows that the GroEL molecule must be much more flexible than previously thought. Characterization of this conformation in terms of tertiary structure will require higher resolution cryo-EM reconstructions. Another obvious question is whether such an expanded state exists in the double ring system. Trypsin digestion experiments (Song et al., 2003) suggest that the heterodimeric substrate used by Chen et al. is protected not only by SR GroEL/ES, but also by the DR system. The expanded state would seem unlikely to occur in the DR system, since all inter-ring contacts would be disrupted. A cryo-EM study of the substrate trapped in the DR system would provide definitive evidence, but it is also obvious that this is a much more difficult task, due to much higher heterogeneity in the population of particles. It would be useful to demonstrate expanded-state cis encapsulation using a large monomeric substrate, in order to simplify interpretation. An important point to remember is that the 86 kDa heterodimeric substrate chosen for this study might be partially folded inside the GroEL cavity, since it is a stable folding intermediate (Wynn et al., 1998). Thus, its volume inside the cavity would be less than for an unfolded substrate of similar mass. Finally, a very difficult problem in cryo-EM reconstruction methods is to differentiate between (1) views of different orientations of the same structure in a homogeneous population and (2) actual structural heterogeneity in a particle population. In this study, the authors knew from the outset that heterogeneity must exist, since some particles would carry substrate and others would not, even without the expectation of two different conformations. They successfully sorted out this mess by starting with initial references that were identical except for the addition of random noise, and continuing with multiple-reference alignment. This appeared to have been enough to allow for separation of the mixed images into more homogeneous subgroups. It is an intriguing and simple tool, and hopefully it can be used generally in other cases of heterogeneous populations of conformations.