Gibberellin promotes plant growth by downregulating DELLA proteins, which act as growth repressors. In the presence of gibberellin, the gibberellin receptor GID1 binds to DELLA proteins, triggering their degradation through polyubiquitination by the SCF^SLY1/GID2 ubiquitin E3 ligase. Despite extensive studies, the molecular mechanisms by which DELLA proteins assemble with SCF^SLY1/GID2 to regulate plant growth remain poorly understood. Here, we present two cryo-electron microscopy structures of the Arabidopsis thaliana DELLA protein RGA in complex with GID1A and GID1A-SLY1-ASK2, respectively. Structural analyses revealed that RGA interacts with GID1A and SLY1 through nonoverlapping binding surfaces, stabilizing the proteins. This suggests that the SCF^SLY1-RGA-GID1A complex assembles through a stepwise stabilization process induced by gibberellin. Furthermore, structural comparison with GRAS proteins indicates that RGA does not interact with IDD family transcription factors when bound to SLY1, suggesting that DELLA protein binding to GID1/SLY1 and to transcription factors is mutually exclusive. These findings provide insights into the gibberellin-mediated regulation of transcription factor activity by DELLA proteins.

Bacillus subtilis SigB is an alternative sigma factor that initiates the transcription of stress-responsive genes. The anti-sigma factor RsbW tightly binds SigB to suppress its activity under normal growth conditions and releases it when nonphosphorylated RsbV binds to RsbW in response to stress signals. To understand the regulation of SigB activity by RsbV and RsbW based on structural features, crystal structures and a small-angle X-ray scattering (SAXS) envelope structure of the RsbV–RsbW complex were determined. The crystal structures showed that RsbV and RsbW form a heterotetramer in a similar manner to a SpoIIAA–SpoIIAB tetramer. Multi-angle light scattering and SAXS revealed that the RsbV–RsbW complex is an octamer in solution. Superimposition of the crystal structure on the SAXS envelope structure showed that the unique dimeric interface of RsbW mediates the formation of an RsbV–RsbW octamer and does not prevent RsbV and SigB from binding to RsbW. These results provide structural insights into the molecular assembly of the RsbV–RsbW complex and the regulation of SigB activity.

The stressosome transduces environmental stress signals to SigB to upregulate SigB-dependent transcription, which is required for bacterial viability. The stressosome core is composed of RsbS and at least one of the RsbR paralogs. A previous cryo-electron microscopy (cryo-EM) structure of the RsbRA–RsbS complex determined under a D2 symmetry restraint showed that the stressosome core forms a pseudo-icosahedron consisting of 60 STAS domains of RsbRA and RsbS. However, it is still unclear how RsbS and one of the RsbR paralogs assemble into the stressosome. Here, an assembly model of the stressosome is presented based on the crystal structure of the RsbS icosahedron and cryo-EM structures of the RsbRA–RsbS complex determined under diverse symmetry restraints (nonsymmetric C1, dihedral D2 and icosahedral I envelopes). 60 monomers of the crystal structure of RsbS fitted well into the I-restrained cryo-EM structure determined at 4.1 Å resolution, even though the STAS domains in the I envelope were averaged. This indicates that RsbS and RsbRA share a highly conserved STAS fold. 22 protrusions observed in the C1 envelope, corresponding to dimers of the RsbRA N-domain, allowed the STAS domains of RsbRA and RsbS to be distinguished in the stressosome core. Based on these, the model of the stressosome core was reconstructed. The mutation of RsbRA residues at the binding interface in the model (R189A/Q191A) significantly reduced the interaction between RsbRA and RsbS. These results suggest that nonconserved residues in the conserved STAS folds between RsbS and RsbR paralogs determine stressosome assembly.

Cryo-electron microscopy (cryo-EM) is a widely used technique for determining macromolecular structures at near-atomic resolution. The theoretical lower limit of particle sizes suitable for cryo-EM structural analysis is estimated to be 38 kDa; typical constraints involve factors such as image contrast and particle alignment accuracy. In this study, we present cryo-EM structures of two protein-ligand complexes near this lower size threshold. First, the structure of the maltose-binding protein complexed with maltose, with a structurally ordered mass of 40.8 kDa, was determined at a resolution of 2.4 Å; both the maltose and water molecules were clearly identified in this structure. The second structure was the kinase domain of human PLK1 complexed with onvansertib, with a structurally ordered mass of 31.6 kDa, below the theoretical 38 kDa limit; this domain was determined at a resolution of 3.4 Å using a gold-supported grid in the presence of β-octyl-glucoside. The density map clearly shows the backbone of PLK1 secondary structure, and the onvansertib. These results demonstrate that cryo-EM can be effectively employed to determine structures of small proteins or domains, and to perform structure-based drug screening for small proteins, without requiring structural fiducials for particle alignment.