Improving NMR Structures of RNA

Bermejo, Guillermo A.; Clore, G. Marius; Schwieters, Charles D.

doi:10.1016/j.str.2016.03.007

Cited by 36 publications

(44 citation statements)

References 42 publications

(90 reference statements)

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“…These results mirror the influence of the new protein-3.1 force field that is used with REPEL, where the new Molprobity atomic radii alleviate clashes and improve the clashscore, as observed for the other proteins in our test test (Fig. 2e) and for nucleic acids (Bermejo et al 2016). In addition, the torsionDB term (Bermejo et al 2012) helps increase the number of favored side chain rotamers.…”

Section: Resultssupporting

confidence: 82%

“…We also examined the performance of the REPEL potential with two versions of the standard Xplor-NIH force field: protein-1.0, which retains many features of the parallhdg.pro and topallhdg. pro parameter and topology files for NMR structure calculations in XPLOR (Brunger 1992), and the newer, now default, version, protein-3.1, where nonbonded parameters have been updated and the atomic radii have been modified to those of Molprobity, to provide a more realistic representation of atomic radii and improve nonbonded repulsions (Bermejo et al 2016). …”

Section: Resultsmentioning

confidence: 99%

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High quality NMR structures: a new force field with implicit water and membrane solvation for Xplor-NIH

et al. 2016

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Structure determination of proteins by NMR is unique in its ability to measure restraints, very accurately, in environments and under conditions that closely mimic those encountered in vivo. For example, advances in solid-state NMR methods enable structure determination of membrane proteins in detergent-free lipid bilayers, and of large soluble proteins prepared by sedimentation, while parallel advances in solution NMR methods and optimization of detergent-free lipid nanodiscs are rapidly pushing the envelope of the size limit for both soluble and membrane proteins. These experimental advantages, however, are partially squandered during structure calculation, because the commonly used force fields are purely repulsive and neglect solvation, Van der Waals forces and electrostatic energy. Here we describe a new force field, and updated energy functions, for protein structure calculations with EEFx implicit solvation, electrostatics, and Van der Waals Lennard-Jones forces, in the widely used program Xplor-NIH. The new force field is based primarily on CHARMM22, facilitating calculations with a wider range of biomolecules. The new EEFx energy function has been rewritten to enable OpenMP parallelism, and optimized to enhance computation efficiency. It implements solvation, electrostatics, and Van der Waals energy terms together, thus ensuring more consistent and efficient computation of the complete nonbonded energy lists. Updates in the related python module allow detailed analysis of the interaction energies and associated parameters. The new force field and energy function work with both soluble proteins and membrane proteins, including those with cofactors or engineered tags, and are very effective in situations where there are sparse experimental restraints. Results obtained for NMR-restrained calculations with a set of five soluble proteins and five membrane proteins show that structures calculated with EEFx have significant improvements in accuracy, precision, and conformation, and that structure refinement can be obtained by short relaxation with EEFx to obtain improvements in these key metrics. These developments broaden the range of biomolecular structures that can be calculated with high fidelity from NMR restraints.

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Section: Resultssupporting

confidence: 82%

Section: Resultsmentioning

confidence: 99%

High quality NMR structures: a new force field with implicit water and membrane solvation for Xplor-NIH

et al. 2016

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“…The structure was recalculated here using Xplor-NIH 2.42, with a modified van der Waal radius of 1.10 (up from 0.9) (61). An initial 100 structures were calculated starting from an extended single strand, using NOE, hydrogen bond, weak planarity (weight = 300.0 for individual base and weight = 6.0 for base pairs), and dihedral angle restraints (21,60).…”

Section: Methodsmentioning

confidence: 99%

Structure and folding of theTetrahymenatelomerase RNA pseudoknot

Cash¹,

Feigon²

2016

Nucleic Acids Res

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Telomerase maintains telomere length at the ends of linear chromosomes using an integral telomerase RNA (TER) and telomerase reverse transcriptase (TERT). An essential part of TER is the template/pseudoknot domain (t/PK) which includes the template, for adding telomeric repeats, template boundary element (TBE), and pseudoknot, enclosed in a circle by stem 1. The Tetrahymena telomerase holoenzyme catalytic core (p65-TER-TERT) was recently modeled in our 9 Å resolution cryo-electron microscopy map by fitting protein and TER domains, including a solution NMR structure of the Tetrahymena pseudoknot. Here, we describe in detail the structure and folding of the isolated pseudoknot, which forms a compact structure with major groove U•A-U and novel C•G-A+ base triples. Base substitutions that disrupt the base triples reduce telomerase activity in vitro. NMR studies also reveal that the pseudoknot does not form in the context of full-length TER in the absence of TERT, due to formation of a competing structure that sequesters pseudoknot residues. The residues around the TBE remain unpaired, potentially providing access by TERT to this high affinity binding site during an early step in TERT-TER assembly. A model for the assembly pathway of the catalytic core is proposed.

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“…The structure of mdP2ab was calculated from an extended unfolded RNA using 802 NOEs and 459 dihedral angle restraints following standard Xplor protocols as described previously (31) but modified to use the updated NIH-Xplor 2.42 (56). The 200 lowest-energy structures were further refined with 95 RDCs.…”

Section: Methodsmentioning

confidence: 99%

Structural conservation in the template/pseudoknot domain of vertebrate telomerase RNA from teleost fish to human

Wang

Yesselman

Zhang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Telomerase is an RNA-protein complex that includes a unique reverse transcriptase that catalyzes the addition of single-stranded telomere DNA repeats onto the 3′ ends of linear chromosomes using an integral telomerase RNA (TR) template. Vertebrate TR contains the template/pseudoknot (t/PK) and CR4/5 domains required for telomerase activity in vitro. All vertebrate pseudoknots include two subdomains: P2ab (helices P2a and P2b with a 5/6-nt internal loop) and the minimal pseudoknot (P2b-P3 and associated loops). A helical extension of P2a, P2a.1, is specific to mammalian TR. Using NMR, we investigated the structures of the full-length TR pseudoknot and isolated subdomains in Oryzias latipes (Japanese medaka fish), which has the smallest vertebrate TR identified to date. We determined the solution NMR structure and studied the dynamics of medaka P2ab, and identified all base pairs and tertiary interactions in the minimal pseudoknot. Despite differences in length and sequence, the structure of medaka P2ab is more similar to human P2ab than predicted, and the medaka minimal pseudoknot has the same tertiary interactions as the human pseudoknot. Significantly, although P2a.1 is not predicted to form in teleost fish, we find that it forms in the full-length pseudoknot via an unexpected hairpin. Model structures of the subdomains are combined to generate a model of t/PK. These results provide evidence that the architecture for the vertebrate t/PK is conserved from teleost fish to human. The organization of the t/PK on telomerase reverse transcriptase for medaka and human is modeled based on the cryoEM structure of Tetrahymena telomerase, providing insight into function.T elomeres are the physical ends of linear chromosomes, composed of telomeric DNA and associated proteins. After each round of cell replication, telomeres shorten because of incomplete 3′-end replication of telomere DNA repeats and nucleolytic processing. Telomerase, a specialized reverse transcriptase, is a large ribonucleoprotein that plays essential roles in protecting the integrity of linear chromosomes in most eukaryotic species (1). Most somatic cells do not contain detectable telomerase activity, and consequently, after 40-60 cell divisions, their telomeres shorten below a critical length [∼12.8 repeats (2)] that leads to replicative senescence or to apoptosis (3). Most cancer cell lines, on the other hand, have significant telomerase activity, which continuously replenishes their telomeres and allows them to divide indefinitely; this dichotomy has made telomerase inhibition an attractive target for the development of anticancer drugs (4, 5). Mutations in human telomerase proteins and RNA cause a number of inherited diseases, such as autosomal dominant dyskeratosis congenita, idiopathic pulmonary fibrosis, myelodysplasia, and aplastic anemia (6-10).The telomerase holoenzyme consists of a unique reverse transcriptase protein (telomerase reverse transcriptase; TERT), which contains the active site for nucleotide addition, an essential telomerase RNA (...

show abstract

Improving NMR Structures of RNA

Cited by 36 publications

References 42 publications

High quality NMR structures: a new force field with implicit water and membrane solvation for Xplor-NIH

High quality NMR structures: a new force field with implicit water and membrane solvation for Xplor-NIH

Structure and folding of theTetrahymenatelomerase RNA pseudoknot

Structural conservation in the template/pseudoknot domain of vertebrate telomerase RNA from teleost fish to human

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