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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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Repeat measurement of distance distributions using independently prepared samples and two different synchrotron X-ray sources.Data for the 10 [A], 25 [B], and 35 [C] base-pair duplexes are shown. Independent samples are labeled A and B followed by the month/year in which they were prepared. The plot key also indicates the synchrotron source (SSRL – Stanford Synchrotron Radiation Laboratory, APS – Advanced Photon Source) followed by the month/year in which the data were collected. The mean and variance of a Gaussian fit to each distribution is reported. The dominant feature of each distribution is extremely reproducible. The smaller variable distribution features appear to correlate with sample preparation and freezer storage time (see sample A in panel A).
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pone-0003229-g004: Repeat measurement of distance distributions using independently prepared samples and two different synchrotron X-ray sources.Data for the 10 [A], 25 [B], and 35 [C] base-pair duplexes are shown. Independent samples are labeled A and B followed by the month/year in which they were prepared. The plot key also indicates the synchrotron source (SSRL – Stanford Synchrotron Radiation Laboratory, APS – Advanced Photon Source) followed by the month/year in which the data were collected. The mean and variance of a Gaussian fit to each distribution is reported. The dominant feature of each distribution is extremely reproducible. The smaller variable distribution features appear to correlate with sample preparation and freezer storage time (see sample A in panel A).

Mentions: We assessed the reproducibility of the scattering interference ruler by repeating distance distribution measurements on independently synthesized samples at two different synchrotron sources (Fig. 4). The samples included end-labeled 10, 25 and 35 base-pair duplexes. We observed small (∼5%) and variable features in the baseline of each distribution that were idiosyncratic to the sample preparation (they likely arise from impurities). However, the dominant probability features were constant. For all three duplexes, the coefficient of variation is less than 1% for the mean of the dominant feature, and less than 7% for the variance. Degradation of the data quality by truncation at low angle and by addition of white noise did not significantly perturb the mean and variance (Fig. S3). Thus, the scattering interference ruler is extremely robust.


A molecular ruler for measuring quantitative distance distributions.

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

Repeat measurement of distance distributions using independently prepared samples and two different synchrotron X-ray sources.Data for the 10 [A], 25 [B], and 35 [C] base-pair duplexes are shown. Independent samples are labeled A and B followed by the month/year in which they were prepared. The plot key also indicates the synchrotron source (SSRL – Stanford Synchrotron Radiation Laboratory, APS – Advanced Photon Source) followed by the month/year in which the data were collected. The mean and variance of a Gaussian fit to each distribution is reported. The dominant feature of each distribution is extremely reproducible. The smaller variable distribution features appear to correlate with sample preparation and freezer storage time (see sample A in panel A).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003229-g004: Repeat measurement of distance distributions using independently prepared samples and two different synchrotron X-ray sources.Data for the 10 [A], 25 [B], and 35 [C] base-pair duplexes are shown. Independent samples are labeled A and B followed by the month/year in which they were prepared. The plot key also indicates the synchrotron source (SSRL – Stanford Synchrotron Radiation Laboratory, APS – Advanced Photon Source) followed by the month/year in which the data were collected. The mean and variance of a Gaussian fit to each distribution is reported. The dominant feature of each distribution is extremely reproducible. The smaller variable distribution features appear to correlate with sample preparation and freezer storage time (see sample A in panel A).
Mentions: We assessed the reproducibility of the scattering interference ruler by repeating distance distribution measurements on independently synthesized samples at two different synchrotron sources (Fig. 4). The samples included end-labeled 10, 25 and 35 base-pair duplexes. We observed small (∼5%) and variable features in the baseline of each distribution that were idiosyncratic to the sample preparation (they likely arise from impurities). However, the dominant probability features were constant. For all three duplexes, the coefficient of variation is less than 1% for the mean of the dominant feature, and less than 7% for the variance. Degradation of the data quality by truncation at low angle and by addition of white noise did not significantly perturb the mean and variance (Fig. S3). Thus, the scattering interference ruler is extremely robust.

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