ATP-dependent proteases - Docking of components in a bacterial complex - Reply

The central issue raised by Ishikawa et al. is that of the configuration of the productive HslVU complex. We note that complexes of HslV and HslU in E. coli are labile and unstable under many conditions1. The original electron microscopy (EM) images of Rohrwild et al.2 appear to show free HslV, free HslU and HslV–HslU complex particles. To explain the discrepancy between the HslV–HslU arrangement in our co-crystals and their negatively stained EM data, we suggested that there might have been a collapse of the fragile I-domain structure in the EM preparations, or a reversal in the orientation of the HslU rings1. Ishikawa et al. interpret their results from cryo-EM at 30 Å resolution in this latter way, using our crystal data of the components. Although these preparations preserve the native structure better than the negatively stained ones, our HslV– HslU samples are also active under crystallization conditions3.