The Rosetta software suite for macromolecular modeling, docking, and design is widely used in pharmaceutical, industrial, academic, non-profit, and government laboratories. Despite its broad modeling capabilities, Rosetta remains consistently among leading software suites when compared to other methods created for highly specialized protein modeling and design tasks. Developed for over two decades by a global community of over 60 laboratories, Rosetta has undergone multiple refactorings, and now comprises over three million lines of code. Here we discuss methods developed in the last five years in Rosetta, involving the latest protocols for structure prediction; protein-protein and protein-small molecule docking; protein structure and interface design; loop modeling; the incorporation of various types of experimental data; modeling of peptides, antibodies and proteins in the immune system, nucleic acids, non-standard chemistries, carbohydrates, and membrane proteins. We briefly discuss improvements to the energy function, user interfaces, and usability of the software. Rosetta is available at www.rosettacommons.org.

Summary Bacteria use rapid contraction of a long sheath of the Type VI secretion system (T6SS) to deliver effectors into a target cell. Here we present an atomic resolution structure of a native contracted Vibrio cholerae sheath determined by cryo-electron microscopy. The sheath subunits, composed of tightly interacting proteins VipA and VipB, assemble into a six-start helix. The helix is stabilized by a core domain assembled from four β-strands donated by one VipA and two VipB molecules. The fold of inner and middle layers is conserved between T6SS and phage sheaths. However, the structure of the outer layer is distinct and suggests a mechanism of interaction of the bacterial sheath with an accessory ATPase, ClpV, that facilitates multiple rounds of effector delivery. Our results provide a mechanistic insight into assembly of contractile nanomachines that bacteria and phages use to translocate macromolecules across membranes.

Summary Mitochondrial inheritance, genome maintenance, and metabolic adaptation depend on organelle fission by Dynamin-Related Protein 1 (DRP1) and its mitochondrial receptors. DRP1 receptors include the paralogs Mitochondrial Dynamics 49 and 51 (MID49/MID51) and Mitochondrial Fission Factor (MFF), but the mechanisms by which these proteins recruit and regulate DRP1 are unknown. Here we present a cryoEM structure of human, full-length DRP1 coassembled with MID49 and an analysis of structure- and disease-based mutations. We report that GTP induces a remarkable elongation and rotation of the G-domain, Bundle-Signaling Element (BSE) and connecting hinge loops of DRP1. In this conformation, a network of multivalent interactions promotes polymerization of a linear DRP1 filament with MID49/MID51. Following coassembly, GTP hydrolysis and exchange lead to MID receptor dissociation, filament shortening and curling of DRP1 oligomers into constricted and closed rings. Together, these views of full-length, receptor- and nucleotide-bound conformations reveal how DRP1 performs mechanical work through nucleotide-driven allostery.