Subject(s)
CRISPR-Cas Systems , Cell- and Tissue-Based Therapy , Gene Editing , Hematopoietic Stem Cell Transplantation , Humans , Gene Editing/methods , CRISPR-Associated Protein 9 , Cell- and Tissue-Based Therapy/adverse effects , Cell- and Tissue-Based Therapy/methods , Antigens, CD34 , Hematopoietic Stem Cells , Hematopoietic Stem Cell Transplantation/methods , Postoperative Complications/genetics , Postoperative Complications/prevention & controlABSTRACT
Structured noncoding RNAs underlie fundamental cellular processes, but determining their three-dimensional structures remains challenging. We demonstrate that integrating ¹H NMR chemical shift data with Rosetta de novo modeling can be used to consistently determine high-resolution RNA structures. On a benchmark set of 23 noncanonical RNA motifs, including 11 'blind' targets, chemical-shift Rosetta for RNA (CS-Rosetta-RNA) recovered experimental structures with high accuracy (0.6-2.0 Å all-heavy-atom r.m.s. deviation) in 18 cases.
Subject(s)
Nuclear Magnetic Resonance, Biomolecular/methods , Nucleotide Motifs , RNA, Untranslated/chemistry , AnimalsABSTRACT
The Rosetta molecular modeling software package provides experimentally tested and rapidly evolving tools for the 3D structure prediction and high-resolution design of proteins, nucleic acids, and a growing number of non-natural polymers. Despite its free availability to academic users and improving documentation, use of Rosetta has largely remained confined to developers and their immediate collaborators due to the code's difficulty of use, the requirement for large computational resources, and the unavailability of servers for most of the Rosetta applications. Here, we present a unified web framework for Rosetta applications called ROSIE (Rosetta Online Server that Includes Everyone). ROSIE provides (a) a common user interface for Rosetta protocols, (b) a stable application programming interface for developers to add additional protocols, (c) a flexible back-end to allow leveraging of computer cluster resources shared by RosettaCommons member institutions, and (d) centralized administration by the RosettaCommons to ensure continuous maintenance. This paper describes the ROSIE server infrastructure, a step-by-step 'serverification' protocol for use by Rosetta developers, and the deployment of the first nine ROSIE applications by six separate developer teams: Docking, RNA de novo, ERRASER, Antibody, Sequence Tolerance, Supercharge, Beta peptide design, NCBB design, and VIP redesign. As illustrated by the number and diversity of these applications, ROSIE offers a general and speedy paradigm for serverification of Rosetta applications that incurs negligible cost to developers and lowers barriers to Rosetta use for the broader biological community. ROSIE is available at http://rosie.rosettacommons.org.
Subject(s)
Internet , Models, Molecular , Software , User-Computer Interface , Molecular Dynamics SimulationABSTRACT
Three-dimensional RNA models fitted into crystallographic density maps exhibit pervasive conformational ambiguities, geometric errors and steric clashes. To address these problems, we present enumerative real-space refinement assisted by electron density under Rosetta (ERRASER), coupled to Python-based hierarchical environment for integrated 'xtallography' (PHENIX) diffraction-based refinement. On 24 data sets, ERRASER automatically corrects the majority of MolProbity-assessed errors, improves the average R(free) factor, resolves functionally important discrepancies in noncanonical structure and refines low-resolution models to better match higher-resolution models.
Subject(s)
Computational Biology , RNA/chemistry , Crystallography, X-Ray , Humans , Models, Molecular , Nucleic Acid Conformation , SoftwareABSTRACT
We report the results of a first, collective, blind experiment in RNA three-dimensional (3D) structure prediction, encompassing three prediction puzzles. The goals are to assess the leading edge of RNA structure prediction techniques; compare existing methods and tools; and evaluate their relative strengths, weaknesses, and limitations in terms of sequence length and structural complexity. The results should give potential users insight into the suitability of available methods for different applications and facilitate efforts in the RNA structure prediction community in ongoing efforts to improve prediction tools. We also report the creation of an automated evaluation pipeline to facilitate the analysis of future RNA structure prediction exercises.
Subject(s)
Nucleic Acid Conformation , RNA/chemistry , Base Sequence , Dimerization , Models, Molecular , Molecular Sequence DataABSTRACT
Atomic-accuracy structure prediction of macromolecules should be achievable by optimizing a physically realistic energy function but is presently precluded by incomplete sampling of a biopolymer's many degrees of freedom. We present herein a working hypothesis, called the "stepwise ansatz," for recursively constructing well-packed atomic-detail models in small steps, enumerating several million conformations for each monomer, and covering all build-up paths. By making use of high-performance computing and the Rosetta framework, we provide first tests of this hypothesis on a benchmark of 15 RNA loop-modeling problems drawn from riboswitches, ribozymes, and the ribosome, including 10 cases that are not solvable by current knowledge-based modeling approaches. For each loop problem, this deterministic stepwise assembly method either reaches atomic accuracy or exposes flaws in Rosetta's all-atom energy function, indicating the resolution of the conformational sampling bottleneck. As a further rigorous test, we have carried out a blind all-atom prediction for a noncanonical RNA motif, the C7.2 tetraloop/receptor, and validated this model through nucleotide-resolution chemical mapping experiments. Stepwise assembly is an enumerative, ab initio build-up method that systematically outperforms existing Monte Carlo and knowledge-based methods for 3D structure prediction.