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A molecular ruler for measuring quantitative distance distributions.

Mathew-Fenn RS, Das R, Silverman JA, Walker PA, Harbury PA - PLoS ONE (2008)

Bottom Line: We demonstrate that measurements with independently prepared samples and using different X-ray sources are highly reproducible, we demonstrate the quantitative accuracy of the first and second moments of the distance distributions, and we demonstrate that the technique recovers complex distribution shapes.Distances measured with the solution scattering-interference ruler match the corresponding crystallographic values, but differ from distances measured previously with alternate ruler techniques.The X-ray scattering interference ruler should be a powerful tool for relating crystal structures to solution structures and for studying molecular fluctuations.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Stanford University, Stanford, California, USA.

ABSTRACT
We report a novel molecular ruler for measurement of distances and distance distributions with accurate external calibration. Using solution X-ray scattering we determine the scattering interference between two gold nanocrystal probes attached site-specifically to a macromolecule of interest. Fourier transformation of the interference pattern provides a model-independent probability distribution for the distances between the probe centers-of-mass. To test the approach, we measure end-to-end distances for a variety of DNA structures. We demonstrate that measurements with independently prepared samples and using different X-ray sources are highly reproducible, we demonstrate the quantitative accuracy of the first and second moments of the distance distributions, and we demonstrate that the technique recovers complex distribution shapes. Distances measured with the solution scattering-interference ruler match the corresponding crystallographic values, but differ from distances measured previously with alternate ruler techniques. The X-ray scattering interference ruler should be a powerful tool for relating crystal structures to solution structures and for studying molecular fluctuations.

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Quantitative accuracy of the first and second moments of the distance distributions.(A) Mean probe-probe separation distances within labeled duplexes is plotted with respect to the number of intervening DNA base-pair steps. Distances measured by X-ray scattering interference, time-resolved single-molecule fluorescence resonance energy transfer (trsmFRET) and double electron-electron spin resonance (DEER) are shown. A three-variable model accounting for rotation of the nanocrystal probes around the helix axis is fit to each data set, giving a fitted helical rise value (Fig. S4). An average rise of 3.32±0.19 Å is observed in naked DNA crystal structures. The scattering interference data fit to a helical rise of 3.29±0.07Å, and a 9Å radial displacement of the nanocrystals off of the helix axis (R2 = 0.999). The trsmFRET data fit to a helical rise of 2.74Å, and 12Å/−2Å radial displacements of the fluorophores off of the helix axis (R2 = 0.999). The DEER data fit to a helical rise of 3.01Å, and a 13Å radial displacement of the spin labels off of the helix axis (R2 = 0.983). The data sets are taken from [24], [33]–[34]. All distances were corrected for the apparent shortening caused by DNA bending (Table 2 in Supplementary Materials S1). (B) The variance in probe-probe separation distance within labeled duplexes is plotted with respect to the number of intervening DNA base-pair steps. The variance grows more rapidly with DNA length in the DEER and the trsmFRET data than in the X-ray scattering data. (C) Comparison of the expected and the observed broadening of a 35 base-pair duplex distance distribution by sub-saturating ethidium bromide. The initial distribution (Black) exhibits a mean of 131.9 Å and a variance of 51 Å2. The distribution in the presence of ethidium (Red) exhibits a mean of 147.7 Å and a variance of 71 Å2. The 15.8 Å shift in the mean corresponds to an average of 4.65 ethidium intercalation events per duplex. Individual duplexes can bind between zero and eight ethidium molecules at pyrimidine-purine base steps. The relative abundance of duplexes with different numbers of bound ethidium molecules is plotted at the left (Black, labeled #EtBr bound distribution). The position of each peak on the horizontal axis corresponds to the increase in duplex length caused by ethidium intercalation. The expected distribution for a 35 base-pair duplex in the presence of ethidium, calculated as the convolution of the distribution in the absence of ethidium with the number of ethidium bound distribution, is shown as a dashed blue line. The expected increase in variance of 22.5 Å2 matches closely to the observed increase of 20 Å2.
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pone-0003229-g005: Quantitative accuracy of the first and second moments of the distance distributions.(A) Mean probe-probe separation distances within labeled duplexes is plotted with respect to the number of intervening DNA base-pair steps. Distances measured by X-ray scattering interference, time-resolved single-molecule fluorescence resonance energy transfer (trsmFRET) and double electron-electron spin resonance (DEER) are shown. A three-variable model accounting for rotation of the nanocrystal probes around the helix axis is fit to each data set, giving a fitted helical rise value (Fig. S4). An average rise of 3.32±0.19 Å is observed in naked DNA crystal structures. The scattering interference data fit to a helical rise of 3.29±0.07Å, and a 9Å radial displacement of the nanocrystals off of the helix axis (R2 = 0.999). The trsmFRET data fit to a helical rise of 2.74Å, and 12Å/−2Å radial displacements of the fluorophores off of the helix axis (R2 = 0.999). The DEER data fit to a helical rise of 3.01Å, and a 13Å radial displacement of the spin labels off of the helix axis (R2 = 0.983). The data sets are taken from [24], [33]–[34]. All distances were corrected for the apparent shortening caused by DNA bending (Table 2 in Supplementary Materials S1). (B) The variance in probe-probe separation distance within labeled duplexes is plotted with respect to the number of intervening DNA base-pair steps. The variance grows more rapidly with DNA length in the DEER and the trsmFRET data than in the X-ray scattering data. (C) Comparison of the expected and the observed broadening of a 35 base-pair duplex distance distribution by sub-saturating ethidium bromide. The initial distribution (Black) exhibits a mean of 131.9 Å and a variance of 51 Å2. The distribution in the presence of ethidium (Red) exhibits a mean of 147.7 Å and a variance of 71 Å2. The 15.8 Å shift in the mean corresponds to an average of 4.65 ethidium intercalation events per duplex. Individual duplexes can bind between zero and eight ethidium molecules at pyrimidine-purine base steps. The relative abundance of duplexes with different numbers of bound ethidium molecules is plotted at the left (Black, labeled #EtBr bound distribution). The position of each peak on the horizontal axis corresponds to the increase in duplex length caused by ethidium intercalation. The expected distribution for a 35 base-pair duplex in the presence of ethidium, calculated as the convolution of the distribution in the absence of ethidium with the number of ethidium bound distribution, is shown as a dashed blue line. The expected increase in variance of 22.5 Å2 matches closely to the observed increase of 20 Å2.

Mentions: Two experiments establish the quantitative accuracy of the scattering interference ruler. First, we used it to determine the helical rise of DNA in solution (reported in [24]). Six end-to-end distance distributions for duplexes between 10 and 35 base-pairs were measured and fit to a three-variable model of the DNA helix (Fig. 5A and Fig. S4). The data indicate a helical rise value of 3.29±0.07 Å, in close agreement with the crystallographic average value of 3.32±0.19 Å [25]. By comparison, identical experiments with two state-of-the-art spectroscopic rulers (a single-molecule fluorescence resonance energy-transfer ruler and a double electron-electron spin resonance ruler) give rise values of 2.74 Å and 3.01 Å respectively. The distance variation measured by the scattering interference ruler is much smaller than the apparent variation measured by spectroscopic rulers (Fig. 5B, Fig. S5).


A molecular ruler for measuring quantitative distance distributions.

Mathew-Fenn RS, Das R, Silverman JA, Walker PA, Harbury PA - PLoS ONE (2008)

Quantitative accuracy of the first and second moments of the distance distributions.(A) Mean probe-probe separation distances within labeled duplexes is plotted with respect to the number of intervening DNA base-pair steps. Distances measured by X-ray scattering interference, time-resolved single-molecule fluorescence resonance energy transfer (trsmFRET) and double electron-electron spin resonance (DEER) are shown. A three-variable model accounting for rotation of the nanocrystal probes around the helix axis is fit to each data set, giving a fitted helical rise value (Fig. S4). An average rise of 3.32±0.19 Å is observed in naked DNA crystal structures. The scattering interference data fit to a helical rise of 3.29±0.07Å, and a 9Å radial displacement of the nanocrystals off of the helix axis (R2 = 0.999). The trsmFRET data fit to a helical rise of 2.74Å, and 12Å/−2Å radial displacements of the fluorophores off of the helix axis (R2 = 0.999). The DEER data fit to a helical rise of 3.01Å, and a 13Å radial displacement of the spin labels off of the helix axis (R2 = 0.983). The data sets are taken from [24], [33]–[34]. All distances were corrected for the apparent shortening caused by DNA bending (Table 2 in Supplementary Materials S1). (B) The variance in probe-probe separation distance within labeled duplexes is plotted with respect to the number of intervening DNA base-pair steps. The variance grows more rapidly with DNA length in the DEER and the trsmFRET data than in the X-ray scattering data. (C) Comparison of the expected and the observed broadening of a 35 base-pair duplex distance distribution by sub-saturating ethidium bromide. The initial distribution (Black) exhibits a mean of 131.9 Å and a variance of 51 Å2. The distribution in the presence of ethidium (Red) exhibits a mean of 147.7 Å and a variance of 71 Å2. The 15.8 Å shift in the mean corresponds to an average of 4.65 ethidium intercalation events per duplex. Individual duplexes can bind between zero and eight ethidium molecules at pyrimidine-purine base steps. The relative abundance of duplexes with different numbers of bound ethidium molecules is plotted at the left (Black, labeled #EtBr bound distribution). The position of each peak on the horizontal axis corresponds to the increase in duplex length caused by ethidium intercalation. The expected distribution for a 35 base-pair duplex in the presence of ethidium, calculated as the convolution of the distribution in the absence of ethidium with the number of ethidium bound distribution, is shown as a dashed blue line. The expected increase in variance of 22.5 Å2 matches closely to the observed increase of 20 Å2.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2566812&req=5

pone-0003229-g005: Quantitative accuracy of the first and second moments of the distance distributions.(A) Mean probe-probe separation distances within labeled duplexes is plotted with respect to the number of intervening DNA base-pair steps. Distances measured by X-ray scattering interference, time-resolved single-molecule fluorescence resonance energy transfer (trsmFRET) and double electron-electron spin resonance (DEER) are shown. A three-variable model accounting for rotation of the nanocrystal probes around the helix axis is fit to each data set, giving a fitted helical rise value (Fig. S4). An average rise of 3.32±0.19 Å is observed in naked DNA crystal structures. The scattering interference data fit to a helical rise of 3.29±0.07Å, and a 9Å radial displacement of the nanocrystals off of the helix axis (R2 = 0.999). The trsmFRET data fit to a helical rise of 2.74Å, and 12Å/−2Å radial displacements of the fluorophores off of the helix axis (R2 = 0.999). The DEER data fit to a helical rise of 3.01Å, and a 13Å radial displacement of the spin labels off of the helix axis (R2 = 0.983). The data sets are taken from [24], [33]–[34]. All distances were corrected for the apparent shortening caused by DNA bending (Table 2 in Supplementary Materials S1). (B) The variance in probe-probe separation distance within labeled duplexes is plotted with respect to the number of intervening DNA base-pair steps. The variance grows more rapidly with DNA length in the DEER and the trsmFRET data than in the X-ray scattering data. (C) Comparison of the expected and the observed broadening of a 35 base-pair duplex distance distribution by sub-saturating ethidium bromide. The initial distribution (Black) exhibits a mean of 131.9 Å and a variance of 51 Å2. The distribution in the presence of ethidium (Red) exhibits a mean of 147.7 Å and a variance of 71 Å2. The 15.8 Å shift in the mean corresponds to an average of 4.65 ethidium intercalation events per duplex. Individual duplexes can bind between zero and eight ethidium molecules at pyrimidine-purine base steps. The relative abundance of duplexes with different numbers of bound ethidium molecules is plotted at the left (Black, labeled #EtBr bound distribution). The position of each peak on the horizontal axis corresponds to the increase in duplex length caused by ethidium intercalation. The expected distribution for a 35 base-pair duplex in the presence of ethidium, calculated as the convolution of the distribution in the absence of ethidium with the number of ethidium bound distribution, is shown as a dashed blue line. The expected increase in variance of 22.5 Å2 matches closely to the observed increase of 20 Å2.
Mentions: Two experiments establish the quantitative accuracy of the scattering interference ruler. First, we used it to determine the helical rise of DNA in solution (reported in [24]). Six end-to-end distance distributions for duplexes between 10 and 35 base-pairs were measured and fit to a three-variable model of the DNA helix (Fig. 5A and Fig. S4). The data indicate a helical rise value of 3.29±0.07 Å, in close agreement with the crystallographic average value of 3.32±0.19 Å [25]. By comparison, identical experiments with two state-of-the-art spectroscopic rulers (a single-molecule fluorescence resonance energy-transfer ruler and a double electron-electron spin resonance ruler) give rise values of 2.74 Å and 3.01 Å respectively. The distance variation measured by the scattering interference ruler is much smaller than the apparent variation measured by spectroscopic rulers (Fig. 5B, Fig. S5).

Bottom Line: We demonstrate that measurements with independently prepared samples and using different X-ray sources are highly reproducible, we demonstrate the quantitative accuracy of the first and second moments of the distance distributions, and we demonstrate that the technique recovers complex distribution shapes.Distances measured with the solution scattering-interference ruler match the corresponding crystallographic values, but differ from distances measured previously with alternate ruler techniques.The X-ray scattering interference ruler should be a powerful tool for relating crystal structures to solution structures and for studying molecular fluctuations.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Stanford University, Stanford, California, USA.

ABSTRACT
We report a novel molecular ruler for measurement of distances and distance distributions with accurate external calibration. Using solution X-ray scattering we determine the scattering interference between two gold nanocrystal probes attached site-specifically to a macromolecule of interest. Fourier transformation of the interference pattern provides a model-independent probability distribution for the distances between the probe centers-of-mass. To test the approach, we measure end-to-end distances for a variety of DNA structures. We demonstrate that measurements with independently prepared samples and using different X-ray sources are highly reproducible, we demonstrate the quantitative accuracy of the first and second moments of the distance distributions, and we demonstrate that the technique recovers complex distribution shapes. Distances measured with the solution scattering-interference ruler match the corresponding crystallographic values, but differ from distances measured previously with alternate ruler techniques. The X-ray scattering interference ruler should be a powerful tool for relating crystal structures to solution structures and for studying molecular fluctuations.

Show MeSH
Related in: MedlinePlus