Stabilizing proteins, simplified: A Rosetta-based webtool for predicting favorable mutations.

Many proteins have low thermodynamic stability, which can lead to low expression yields and limit functionality in research, industrial and clinical settings. This article introduces two, web-based tools that use the high-resolution structure of a protein along with the Rosetta molecular modeling program to predict stabilizing mutations. The protocols were recently applied to three genetically and structurally distinct proteins and successfully predicted mutations that improved thermal stability and/or protein yield.

PyRosetta Jupyter Notebooks Teach Biomolecular Structure Prediction and Design.

Biomolecular structure drives function, and computational capabilities have progressed such that the prediction and computational design of biomolecular structures is increasingly feasible. Because computational biophysics attracts students from many different backgrounds and with different levels of resources, teaching the subject can be challenging. One strategy to teach diverse learners is with interactive multimedia material that promotes self-paced, active learning.

Comparative Analysis of Sulfonium-π, Ammonium-π, and Sulfur-π Interactions and Relevance to SAM-Dependent Methyltransferases.

We report the measurement and analysis of sulfonium-π, thioether-π, and ammonium-π interactions in a β-hairpin peptide model system, coupled with computational investigation and PDB analysis. These studies indicated that the sulfonium-π interaction is the strongest and that polarizability contributes to the stronger interaction with sulfonium relative to ammonium. Computational studies demonstrate that differences in solvation of the trimethylsulfonium versus the trimethylammonium group also contribute to the stronger sulfonium-π interaction.

Ensuring scientific reproducibility in bio-macromolecular modeling via extensive, automated benchmarks.

Each year vast international resources are wasted on irreproducible research. The scientific community has been slow to adopt standard software engineering practices, despite the increases in high-dimensional data, complexities of workflows, and computational environments. Here we show how scientific software applications can be created in a reproducible manner when simple design goals for reproducibility are met.

Macromolecular modeling and design in Rosetta: recent methods and frameworks.

The Rosetta software for macromolecular modeling, docking and design is extensively used in laboratories worldwide. During two decades of development by a community of laboratories at more than 60 institutions, Rosetta has been continuously refactored and extended. Its advantages are its performance and interoperability between broad modeling capabilities.

Better together: Elements of successful scientific software development in a distributed collaborative community.

Many scientific disciplines rely on computational methods for data analysis, model generation, and prediction. Implementing these methods is often accomplished by researchers with domain expertise but without formal training in software engineering or computer science. This arrangement has led to underappreciation of sustainability and maintainability of scientific software tools developed in academic environments.

A cyber-linked undergraduate research experience in computational biomolecular structure prediction and design.

Computational biology is an interdisciplinary field, and many computational biology research projects involve distributed teams of scientists. To accomplish their work, these teams must overcome both disciplinary and geographic barriers. Introducing new training paradigms is one way to facilitate research progress in computational biology.

Computational design of a specific heavy chain/κ light chain interface for expressing fully IgG bispecific antibodies.

The use of bispecific antibodies (BsAbs) to treat human diseases is on the rise. Increasingly complex and powerful therapeutic mechanisms made possible by BsAbs are spurring innovation of novel BsAb formats and methods for their production. The long-lived in vivo pharmacokinetics, optimal biophysical properties and potential effector functions of natural IgG monoclonal (and monospecific) antibodies has resulted in a push to generate fully IgG BsAb formats with the same quaternary structure as monoclonal IgGs.

Foldit Standalone: a video game-derived protein structure manipulation interface using Rosetta.

Summary: Foldit Standalone is an interactive graphical interface to the Rosetta molecular modeling package. In contrast to most command-line or batch interactions with Rosetta, Foldit Standalone is designed to allow easy, real-time, direct manipulation of protein structures, while also giving access to the extensive power of Rosetta computations. Derived from the user interface of the scientific discovery game Foldit (itself based on Rosetta), Foldit Standalone has added more advanced features and removed the competitive game elements.

The Rosetta All-Atom Energy Function for Macromolecular Modeling and Design.

Over the past decade, the Rosetta biomolecular modeling suite has informed diverse biological questions and engineering challenges ranging from interpretation of low-resolution structural data to design of nanomaterials, protein therapeutics, and vaccines. Central to Rosetta's success is the energy function: a model parametrized from small-molecule and X-ray crystal structure data used to approximate the energy associated with each biomolecule conformation. This paper describes the mathematical models and physical concepts that underlie the latest Rosetta energy function, called the Rosetta Energy Function 2015 (REF15).

Computationally Designed Bispecific Antibodies using Negative State Repertoires.

A challenge in the structure-based design of specificity is modeling the negative states, i.e., the complexes that you do not want to form.

Combined covalent-electrostatic model of hydrogen bonding improves structure prediction with Rosetta.

Interactions between polar atoms are challenging to model because at very short ranges they form hydrogen bonds (H-bonds) that are partially covalent in character and exhibit strong orientation preferences; at longer ranges the orientation preferences are lost, but significant electrostatic interactions between charged and partially charged atoms remain. To simultaneously model these two types of behavior, we refined an orientation dependent model of hydrogen bonds [Kortemme et al. J.

SwiftLib: rapid degenerate-codon-library optimization through dynamic programming.

Degenerate codon (DC) libraries efficiently address the experimental library-size limitations of directed evolution by focusing diversity toward the positions and toward the amino acids (AAs) that are most likely to generate hits; however, manually constructing DC libraries is challenging, error prone and time consuming. This paper provides a dynamic programming solution to the task of finding the best DCs while keeping the size of the library beneath some given limit, improving on the existing integer-linear programming formulation. It then extends the algorithm to consider multiple DCs at each position, a heretofore unsolved problem, while adhering to a constraint on the number of primers needed to synthesize the library.

Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface.

Robust generation of IgG bispecific antibodies has been a long-standing challenge. Existing methods require extensive engineering of each individual antibody, discovery of common light chains, or complex and laborious biochemical processing. Here we combine computational and rational design approaches with experimental structural validation to generate antibody heavy and light chains with orthogonal Fab interfaces.

Scientific benchmarks for guiding macromolecular energy function improvement.

Accurate energy functions are critical to macromolecular modeling and design. We describe new tools for identifying inaccuracies in energy functions and guiding their improvement, and illustrate the application of these tools to the improvement of the Rosetta energy function. The feature analysis tool identifies discrepancies between structures deposited in the PDB and low-energy structures generated by Rosetta; these likely arise from inaccuracies in the energy function.

Computational protein design with explicit consideration of surface hydrophobic patches.

De novo protein design requires the identification of amino-acid sequences that favor the target-folded conformation and are soluble in water. One strategy for promoting solubility is to disallow hydrophobic residues on the protein surface during design. However, naturally occurring proteins often have hydrophobic amino acids on their surface that contribute to protein stability via the partial burial of hydrophobic surface area or play a key role in the formation of protein-protein interactions.

A generic program for multistate protein design.

Some protein design tasks cannot be modeled by the traditional single state design strategy of finding a sequence that is optimal for a single fixed backbone. Such cases require multistate design, where a single sequence is threaded onto multiple backbones (states) and evaluated for its strengths and weaknesses on each backbone. For example, to design a protein that can switch between two specific conformations, it is necessary to to find a sequence that is compatible with both backbone conformations.

Modeling symmetric macromolecular structures in Rosetta3.

Symmetric protein assemblies play important roles in many biochemical processes. However, the large size of such systems is challenging for traditional structure modeling methods. This paper describes the implementation of a general framework for modeling arbitrary symmetric systems in Rosetta3.

RosettaScripts: a scripting language interface to the Rosetta macromolecular modeling suite.

Macromolecular modeling and design are increasingly useful in basic research, biotechnology, and teaching. However, the absence of a user-friendly modeling framework that provides access to a wide range of modeling capabilities is hampering the wider adoption of computational methods by non-experts. RosettaScripts is an XML-like language for specifying modeling tasks in the Rosetta framework.

De novo enzyme design using Rosetta3.

The Rosetta de novo enzyme design protocol has been used to design enzyme catalysts for a variety of chemical reactions, and in principle can be applied to any arbitrary chemical reaction of interest. The process has four stages: 1) choice of a catalytic mechanism and corresponding minimal model active site, 2) identification of sites in a set of scaffold proteins where this minimal active site can be realized, 3) optimization of the identities of the surrounding residues for stabilizing interactions with the transition state and primary catalytic residues, and 4) evaluation and ranking the resulting designed sequences. Stages two through four of this process can be carried out with the Rosetta package, while stage one needs to be done externally.

Structure-guided forcefield optimization.

Accurate modeling of biomolecular systems requires accurate forcefields. Widely used molecular mechanics (MM) forcefields obtain parameters from experimental data and quantum chemistry calculations on small molecules but do not have a clear way to take advantage of the information in high-resolution macromolecular structures. In contrast, knowledge-based methods largely ignore the physical chemistry of interatomic interactions, and instead derive parameters almost exclusively from macromolecular structures.

Role of conformational sampling in computing mutation-induced changes in protein structure and stability.

The prediction of changes in protein stability and structure resulting from single amino acid substitutions is both a fundamental test of macromolecular modeling methodology and an important current problem as high throughput sequencing reveals sequence polymorphisms at an increasing rate. In principle, given the structure of a wild-type protein and a point mutation whose effects are to be predicted, an accurate method should recapitulate both the structural changes and the change in the folding-free energy. Here, we explore the performance of protocols which sample an increasing diversity of conformations.

ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules.

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.