A toolkit and benchmark study for FRET-restrained high-precision structural modeling

Lehrstuhl für Molekulare Physikalische Chemie, Heinrich-Heine-Universität (HHU), Düsseldorf, Germany.
Nature Methods (Impact Factor: 32.07). 11/2012; 9(12). DOI: 10.1038/nmeth.2222
Source: PubMed


We present a comprehensive toolkit for Förster resonance energy transfer (FRET)-restrained modeling of biomolecules and their complexes for quantitative applications in structural biology. A dramatic improvement in the precision of FRET-derived structures is achieved by explicitly considering spatial distributions of dye positions, which greatly reduces uncertainties due to flexible dye linkers. The precision and confidence levels of the models are calculated by rigorous error estimation. The accuracy of this approach is demonstrated by docking a DNA primer-template to HIV-1 reverse transcriptase. The derived model agrees with the known X-ray structure with an r.m.s. deviation of 0.5 Å. Furthermore, we introduce FRET-guided 'screening' of a large structural ensemble created by molecular dynamics simulations. We used this hybrid approach to determine the formerly unknown configuration of the flexible single-strand template overhang.

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Available from: Paul J Rothwell, Jan 29, 2014
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    • "(c) Modelling of the ternary Fen1:DNA:PCNA complex using MD simulation derived from (26) and the FRET distances extracted in (b). Pink and cyan spheres represent mean dye positions modelled using the AV approach (27,37) (See ‘Materials and Methods’ section and Supplementary Material for details). "
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    ABSTRACT: Flap endonuclease 1 (Fen1) is a highly conserved structure-specific nuclease that catalyses a specific incision to remove 5′ flaps in double-stranded DNA substrates. Fen1 plays an essential role in key cellular processes, such as DNA replication and repair, and mutations that compromise Fen1 expression levels or activity have severe health implications in humans. The nuclease activity of Fen1 and other FEN family members can be stimulated by processivity clamps such as proliferating cell nuclear antigen (PCNA); however, the exact mechanism of PCNA activation is currently unknown. Here, we have used a combination of ensemble and single-molecule Förster resonance energy transfer together with protein-induced fluorescence enhancement to uncouple and investigate the substrate recognition and catalytic steps of Fen1 and Fen1/PCNA complexes. We propose a model in which upon Fen1 binding, a highly dynamic substrate is bent and locked into an open flap conformation where specific Fen1/DNA interactions can be established. PCNA enhances Fen1 recognition of the DNA substrate by further promoting the open flap conformation in a step that may involve facilitated threading of the 5′ ssDNA flap. Merging our data with existing crystallographic and molecular dynamics simulations we provide a solution-based model for the Fen1/PCNA/DNA ternary complex.
    Full-text · Article · Nov 2013 · Nucleic Acids Research
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    • "To this end, we encourage researchers to submit their ensembles and the corresponding primary experimental data. We will also consider including additional types of data, such as fluorescence resonance energy transfer (FRET) data, which might rapidly gain importance in determining dynamic structures (50). The database already holds unfolded ensemble(s) of globular proteins (29), which may lead to a better understanding of protein folding, and also address the question as to whether IDPs are fundamentally different from denatured states of folded proteins [cf. the term ‘natively denatured proteins’ for IDPs (51)]. "
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    ABSTRACT: The goal of pE-DB ( is to serve as an openly accessible database for the deposition of structural ensembles of intrinsically disordered proteins (IDPs) and of denatured proteins based on nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and other data measured in solution. Owing to the inherent flexibility of IDPs, solution techniques are particularly appropriate for characterizing their biophysical properties, and structural ensembles in agreement with these data provide a convenient tool for describing the underlying conformational sampling. Database entries consist of (i) primary experimental data with descriptions of the acquisition methods and algorithms used for the ensemble calculations, and (ii) the structural ensembles consistent with these data, provided as a set of models in a Protein Data Bank format. PE-DB is open for submissions from the community, and is intended as a forum for disseminating the structural ensembles and the methodologies used to generate them. While the need to represent the IDP structures is clear, methods for determining and evaluating the structural ensembles are still evolving. The availability of the pE-DB database is expected to promote the development of new modeling methods and leads to a better understanding of how function arises from disordered states.
    Full-text · Article · Oct 2013 · Nucleic Acids Research
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    • "In this article, the HIV-1 5 0 UTR RNA structure has been predicted from a homogenized monomeric population. Although the accuracy of SmFRET distance measurements can be affected by several different variables such as dye orientation, mobility and shot noise, it has been shown previously (Kalinin et al., 2012) that higher resolution structures can be achieved from low-precision FRET values. The details are driven by the force field of the molecular dynamic prediction but only after the area of structure space is selected by FRET restraints. "
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    ABSTRACT: HIV-1 genomic RNA has a noncoding 5' region containing sequential conserved structural motifs that control many parts of the life cycle. Very limited data exist on their three-dimensional (3D) conformation and, hence, how they work structurally. To assemble a working model, we experimentally reassessed secondary structure elements of a 240-nt region and used single-molecule distances, derived from fluorescence resonance energy transfer, between defined locations in these elements as restraints to drive folding of the secondary structure into a 3D model with an estimated resolution below 10 Å. The folded 3D model satisfying the data is consensual with short nuclear-magnetic-resonance-solved regions and reveals previously unpredicted motifs, offering insight into earlier functional assays. It is a 3D representation of this entire region, with implications for RNA dimerization and protein binding during regulatory steps. The structural information of this highly conserved region of the virus has the potential to reveal promising therapeutic targets.
    Full-text · Article · May 2013 · Structure
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