We have recently completed a full re-architecturing of the Rosetta molecular modeling program, generalizing and expanding its existing functionality. The new architecture enables the rapid prototyping of novel protocols by providing easy to use interfaces to powerful tools for molecular modeling. The source code of this rearchitecturing has been released as Rosetta3 and is freely available for academic use. At the time of its release, it contained 470,000 lines of code. Counting currently unpublished protocols at the time of this writing, the source includes 1,285,000 lines. Its rapid growth is a testament to its ease of use. This document describes the requirements for our new architecture, justifies the design decisions, sketches out central classes, and highlights a few of the common tasks that the new software can perform.

Proteins exploit the conformational variability of loop regions to carry out diverse biological tasks including molecular recognition and signal transduction. New algorithms to engineer these functions by combining loop building and sequence design therefore have enormous practical applications but require high-resolution 'loop reconstruction': the modeling of protein loop conformations, given the amino acid sequence. Loop reconstruction in protein design may be simplified conceptually by restricting changes to the functional loop regions. However, despite progress in loop prediction methods 1,2 , design applications are limited by the difficulty in modeling purely local conformational moves and by the need for advances in sampling and evaluating loop conformations.Here we address these challenges with a robotics-inspired local loop reconstruction method for peptide chains, called kinematic closure (KIC). Calculating the accessible conformations of objects subject to constraints, such as determining the possible positions of the interior joints of a robot arm given fixed positions for the shoulder and fingertips, has been well-studied in inverse kinematics, a subfield of robotics. Building on the first 3 and subsequent applications (Supplementary Methods) of kinematics to protein modeling, the KIC method presented here analytically determines all mechanically accessible conformations for 6 torsions of a peptide chain of any length, while simultaneously sampling the remaining torsions and N-Cα-C bond angles using polynomial resultants 4 (Fig. 1a, Supplementary Methods and Supplementary Fig. 1). To enable a range of applications, we coupled KIC to the Rosetta method for protein structure modeling 5 . Our loop reconstruction protocol iterates KIC calculations as Monte Carlo moves first with loop backbone minimization in a low-resolution stage, in which side-chains are represented as centroids, and then in a high-resolution all-atom stage with minimization of the loop backbone and all side chains in the loop environment ( Supplementary Fig. 2 and Supplementary Methods). At the beginning of each KIC simulation, we discard all native loop bond lengths, bond angles and torsions. In addition, we perform reconstructions without knowledge of native side-chain conformations in both the loop and the protein scaffold (Supplementary Methods), which makes prediction substantially more challenging but We found that KIC substantially improves model accuracy over the standard loop building method in Rosetta, which combines insertion of torsion segments from homologous proteins and a numerical closure technique6. We generated 1,000 models by the KIC method and compared its performance to the standard Rosetta method with the same number of Monte Carlo steps on twenty-five 12-residue protein loops (dataset 1; ref. 7). For each protein, we computed the root mean squared (r.m.s.) deviation of the backbone atoms of the best scoring loop model to the crystallographic loop, after superimposing the non-loop regions of the model onto the ...

Genetically modified organisms (GMOs) are increasingly deployed at large scales and in open environments. Genetic biocontainment strategies are needed to prevent unintended proliferation of GMOs in natural ecosystems. Existing biocontainment methods are insufficient either because they impose evolutionary pressure on the organism to eject the safeguard, because they can be circumvented by environmentally available compounds, or because they can be overcome by horizontal gene transfer (HGT). Here we computationally redesign essential enzymes in the first organism possessing an altered genetic code to confer metabolic dependence on nonstandard amino acids for survival. The resulting GMOs cannot metabolically circumvent their biocontainment mechanisms using environmentally available compounds, and they exhibit unprecedented resistance to evolutionary escape via mutagenesis and HGT. This work provides a foundation for safer GMOs that are isolated from natural ecosystems by reliance on synthetic metabolites.

Summary Calcium/calmodulin-dependent kinase II (CaMKII) forms a highly conserved dodecameric assembly that is sensitive to the frequency of calcium pulse trains. Neither the structure of the dodecameric assembly nor how it regulates CaMKII are known. We present the crystal structure of an autoinhibited full-length human CaMKII holoenzyme, revealing an unexpected compact arrangement of kinase domains docked against a central hub, with the calmodulin binding sites completely inaccessible. We show that this compact docking is important for the autoinhibition of the kinase domains and for setting the calcium response of the holoenzyme. Comparison of CaMKII isoforms, which differ in the length of the linker between the kinase domain and the hub, demonstrates that these interactions can be strengthened or weakened by changes in linker length. This equilibrium between autoinhibited states provides a simple mechanism for tuning the calcium response without changes in either the hub or the kinase domains.