33 research outputs found
Correcting pervasive errors in RNA crystallography through enumerative structure prediction
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 Rfree factor,
resolves functionally important discrepancies in noncanonical structure and
refines low-resolution models to better match higher-resolution models
Serverification of Molecular Modeling Applications: the Rosetta Online Server that Includes Everyone (ROSIE)
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
Massively Parallel RNA Chemical Mapping with a Reduced Bias MAP-seq Protocol
Chemical mapping methods probe RNA structure by revealing and leveraging
correlations of a nucleotide's structural accessibility or flexibility with its
reactivity to various chemical probes. Pioneering work by Lucks and colleagues
has expanded this method to probe hundreds of molecules at once on an Illumina
sequencing platform, obviating the use of slab gels or capillary
electrophoresis on one molecule at a time. Here, we describe optimizations to
this method from our lab, resulting in the MAP-seq protocol (Multiplexed
Accessibility Probing read out through sequencing), version 1.0. The protocol
permits the quantitative probing of thousands of RNAs at once, by several
chemical modification reagents, on the time scale of a day using a table-top
Illumina machine. This method and a software package MAPseeker
(http://simtk.org/home/map_seeker) address several potential sources of bias,
by eliminating PCR steps, improving ligation efficiencies of ssDNA adapters,
and avoiding problematic heuristics in prior algorithms. We hope that the
step-by-step description of MAP-seq 1.0 will help other RNA mapping
laboratories to transition from electrophoretic to next-generation sequencing
methods and to further reduce the turnaround time and any remaining biases of
the protocol.Comment: 22 pages, 5 figure
Atomic-accuracy prediction of protein loop structures through an RNA-inspired ansatz
Consistently predicting biopolymer structure at atomic resolution from
sequence alone remains a difficult problem, even for small sub-segments of
large proteins. Such loop prediction challenges, which arise frequently in
comparative modeling and protein design, can become intractable as loop lengths
exceed 10 residues and if surrounding side-chain conformations are erased. This
article introduces a modeling strategy based on a 'stepwise ansatz', recently
developed for RNA modeling, which posits that any realistic all-atom molecular
conformation can be built up by residue-by-residue stepwise enumeration. When
harnessed to a dynamic-programming-like recursion in the Rosetta framework, the
resulting stepwise assembly (SWA) protocol enables enumerative sampling of a 12
residue loop at a significant but achievable cost of thousands of CPU-hours. In
a previously established benchmark, SWA recovers crystallographic conformations
with sub-Angstrom accuracy for 19 of 20 loops, compared to 14 of 20 by KIC
modeling with a comparable expenditure of computational power. Furthermore, SWA
gives high accuracy results on an additional set of 15 loops highlighted in the
biological literature for their irregularity or unusual length. Successes
include cis-Pro touch turns, loops that pass through tunnels of other
side-chains, and loops of lengths up to 24 residues. Remaining problem cases
are traced to inaccuracies in the Rosetta all-atom energy function. In five
additional blind tests, SWA achieves sub-Angstrom accuracy models, including
the first such success in a protein/RNA binding interface, the YbxF/kink-turn
interaction in the fourth RNA-puzzle competition. These results establish
all-atom enumeration as a systematic approach to protein structure that can
leverage high performance computing and physically realistic energy functions
to more consistently achieve atomic resolution.Comment: Identity of four-loop blind test protein and parts of figures 5 have
been omitted in this preprint to ensure confidentiality of the protein
structure prior to its public releas
Recommended from our members
Serverification of Molecular Modeling Applications: The Rosetta Online Server That Includes Everyone (ROSIE)
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 fre
Recommended from our members
Serverification of Molecular Modeling Applications: The Rosetta Online Server That Includes Everyone (ROSIE)
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 fre
Structure determination of noncanonical RNA motifs guided by 1 H NMR chemical shifts
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 Ă
all-heavy-atom r.m.s. deviation) in 18 cases. © 2014 Nature America, Inc. All rights reserved
Structure determination of noncanonical RNA motifs guided by 1 H NMR chemical shifts
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 Ă
all-heavy-atom r.m.s. deviation) in 18 cases. © 2014 Nature America, Inc. All rights reserved
An enumerative stepwise ansatz enables atomic-accuracy RNA loop modeling
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 biopolymerâs many degrees of freedom. We present herein a working hypothesis, called the âstepwise ansatz,â 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 Rosettaâs 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
Recommended from our members
Serverification of molecular modeling applications: the Rosetta Online Server that Includes Everyone (ROSIE).
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 codes 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