Reconstructing three-dimensional shape envelopes from time-resolved small-angle X-ray scattering data

Lamb, Jessica S.; Kwok, Lisa W.; Qiu, Xiangyun; Andresen, Kurt; Park, Hye Yoon; Pollack, Lois

doi:10.1107/s0021889808028264

Cited by 24 publications

(18 citation statements)

References 25 publications

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“…The sizes and shapes deduced from analyses of cryo-EM images are in agreement with structures we obtain independently from 3D reconstructions of SAXS form factors (Konarev et al 2006;Lipfert et al 2007a;Lamb et al 2008) and those we predict by molecular dynamics (MD) software recently developed (Jonikas et al 2009) for simulating spatial conformations of RNA secondary structures. With this combination of approaches, we demonstrate that the 3D anisotropy of long ssRNAs can be directly deduced from analysis of their cryo-EM images.…”

Section: Introductionsupporting

confidence: 65%

“…Whereas high-field nuclear magnetic resonance (NMR) can directly and reliably probe the structures of short ssRNAs in solution (Flinders and Dieckmann 2006), this technique cannot be used for molecules longer than 1000 nt because of their slow rotational diffusion. Third, while low-resolution three-dimensional (3D) reconstructions of RNAs in solution can be obtained from small-angle X-ray scattering (SAXS) reconstructions, the method has generally been applied to RNA molecules z200 nt in length or shorter, often containing one dominant secondary structure (Lipfert et al 2007a(Lipfert et al ,b, 2008Lamb et al 2008). Applying this technique to larger RNAs is considerably more difficult because the scattered intensity relevant for overall size determination lies at very small angles, close to the incident beam.…”

Section: Introductionmentioning

confidence: 99%

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Visualizing large RNA molecules in solution

Gopal¹,

Zhou²,

Knobler³

et al. 2011

RNA

101

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Single-stranded RNAs (ssRNAs) longer than a few hundred nucleotides do not have a unique structure in solution. Their equilibrium properties therefore reflect the average of an ensemble of structures. We use cryo-electron microscopy to image projections of individual long ssRNA molecules and characterize the anisotropy of their ensembles in solution. A flattened prolate volume is found to best represent the shapes of these ensembles. The measured sizes and anisotropies are in good agreement with complementary determinations using small-angle X-ray scattering and coarse-grained molecular dynamics simulations. A long viral ssRNA is compared with shorter noncoding transcripts to demonstrate that prolate geometry and flatness are generic properties independent of sequence length and origin. The anisotropy persists under physiological as well as low-ionic-strength conditions, revealing a direct correlation between secondary structure asymmetry and 3D shape and size. We discuss the physical origin of the generic anisotropy and its biological implications.

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Section: Introductionsupporting

confidence: 65%

Section: Introductionmentioning

confidence: 99%

Visualizing large RNA molecules in solution

Gopal¹,

Zhou²,

Knobler³

et al. 2011

RNA

101

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“…The folding of P4-P6 has been followed in multiple studies, with folding rate constants of 2-30 s −1 observed across a range of conditions (52)(53)(54)(55)(61)(62)(63)(64)(65). However, interpretation of these overall rate constants has been stymied by a lack of knowledge of which individual folding steps were followed and rate limiting.…”

Section: Resultsmentioning

confidence: 99%

“…The control afforded by this well-behaved, established system provides an opportunity to deepen understanding and develop and test generalizable principles beyond this model system, provided that a detailed framework is in place to provide context for interpretation. Here, we use single-molecule FRET (smFRET) with a series of P4-P6 mutants to isolate and measure specific folding transitions, extending beyond measurements of overall folding rates (52)(53)(54)(55)(61)(62)(63)(64)(65) and constructing a kinetic and thermodynamic framework for P4-P6 RNA. This framework defines a preferred folding pathway, allows this pathway and the partitioning of states to be interpreted in structural terms, helps to uncover the origin of the effects of mutations on folding rates and pathways, and suggests a generalized framework for considering RNA tertiary folding.…”

mentioning

confidence: 99%

Kinetic and thermodynamic framework for P4-P6 RNA reveals tertiary motif modularity and modulation of the folding preferred pathway

Bisaria

Greenfeld

Limouse

et al. 2016

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

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The past decade has seen a wealth of 3D structural information about complex structured RNAs and identification of functional intermediates. Nevertheless, developing a complete and predictive understanding of the folding and function of these RNAs in biology will require connection of individual rate and equilibrium constants to structural changes that occur in individual folding steps and further relating these steps to the properties and behavior of isolated, simplified systems. To accomplish these goals we used the considerable structural knowledge of the folded, unfolded, and intermediate states of P4-P6 RNA. We enumerated structural states and possible folding transitions and determined rate and equilibrium constants for the transitions between these states using single-molecule FRET with a series of mutant P4-P6 variants. Comparisons with simplified constructs containing an isolated tertiary contact suggest that a given tertiary interaction has a stereotyped rate for breaking that may help identify structural transitions within complex RNAs and simplify the prediction of folding kinetics and thermodynamics for structured RNAs from their parts. The preferred folding pathway involves initial formation of the proximal tertiary contact. However, this preference was only ∼10 fold and could be reversed by a single point mutation, indicating that a model akin to a protein-folding contact order model will not suffice to describe RNA folding. Instead, our results suggest a strong analogy with a modified RNA diffusion-collision model in which tertiary elements within preformed secondary structures collide, with the success of these collisions dependent on whether the tertiary elements are in their rare binding-competent conformations.RNA folding | single-molecule FRET | kinetics | folding pathways | RNA tertiary motifs S tructured RNAs play central roles in biology, in the splicing and alternative splicing of eukaryotic pre-mRNAs, in the synthesis of proteins and their transport, and in chromosome maintenance (1-4). Beyond the RNAs and RNA-protein machines involved in these processes, it has been increasingly recognized that Nature has taken extensive advantage of RNA at multiple levels of gene regulation, and considerable current efforts are focused on uncovering the pathways and molecular mechanisms that underlie the functions of small RNAs and long noncoding RNAs (2, 5-8). The pervasive functions of RNA in biology underscore the importance of understanding RNA's fundamental physical properties and, ultimately, of using this understanding to predict the kinetics and thermodynamics of folding and conformational transitions responsible for RNA function.Decades of characterization of RNA folding and structure have led to generalizable principles and provided biological insights. The observations that RNA encodes genetic information, forms highly stable local structures, and can catalyze reactions provided support for the possibility that early life evolved using functional RNAs (9-12). This high stability was also re...

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“…To evaluate the quality of captured scattering profiles the technique of Lamb et al (2008) was followed to calculate the signal-to-noise ratio (S/N) as…”

Section: Scattering Measurementsmentioning

confidence: 99%

Small-angle solution scattering using the mixed-mode pixel array detector

Koerner

Gillilan

Green

et al. 2010

J Synchrotron Radiat

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Solution small-angle X-ray scattering (SAXS) measurements were obtained using a 128 Â 128 pixel X-ray mixed-mode pixel array detector (MMPAD) with an 860 ms readout time. The MMPAD offers advantages for SAXS experiments: a pixel full-well of >2 Â 10 7 10 keV X-rays, a maximum flux rate of 10 8 X-rays pixel À1 s À1, and a sub-pixel point-spread function. Data from the MMPAD were quantitatively compared with data from a charge-coupled device (CCD) fiberoptically coupled to a phosphor screen. MMPAD solution SAXS data from lysozyme solutions were of equal or better quality than data captured by the CCD. The read-noise (normalized by pixel area) of the MMPAD was less than that of the CCD by an average factor of 3.0. Short sample-to-detector distances were required owing to the small MMPAD area (19.2 mm Â 19.2 mm), and were revealed to be advantageous with respect to detector read-noise. As predicted by the Shannon sampling theory and confirmed by the acquisition of lysozyme solution SAXS curves, the MMPAD at short distances is capable of sufficiently sampling a solution SAXS curve for protein shape analysis. The readout speed of the MMPAD was demonstrated by continuously monitoring lysozyme sample evolution as radiation damage accumulated. These experiments prove that a small suitably configured MMPAD is appropriate for time-resolved solution scattering measurements.

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Reconstructing three-dimensional shape envelopes from time-resolved small-angle X-ray scattering data

Cited by 24 publications

References 25 publications

Visualizing large RNA molecules in solution

Visualizing large RNA molecules in solution

Kinetic and thermodynamic framework for P4-P6 RNA reveals tertiary motif modularity and modulation of the folding preferred pathway

Small-angle solution scattering using the mixed-mode pixel array detector

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