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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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Characterization of thioglucose passivated gold nanocrystals.(A) Five superimposed gold autoscattering frames measured in the presence of radical scavengers (top) are vertically offset from five autoscattering frames collected in the absence of radical scavengers (bottom). The frames were collected at one second intervals. Tris (70 mM) and ascorbic acid (10 mM) were used to suppress radiation damage. (B) Gold nanocrystal radius distributions measured for commercially available Nanogold (Red) and three independent thioglucose nanocrystal preparations (labeled with the month/year in which they were prepared). (C) Negative ion matrix assisted laser desorption/ionization mass spectrum of the thioglucose nanocrystal. The inset shows the m/z range centered at 15,500.
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pone-0003229-g001: Characterization of thioglucose passivated gold nanocrystals.(A) Five superimposed gold autoscattering frames measured in the presence of radical scavengers (top) are vertically offset from five autoscattering frames collected in the absence of radical scavengers (bottom). The frames were collected at one second intervals. Tris (70 mM) and ascorbic acid (10 mM) were used to suppress radiation damage. (B) Gold nanocrystal radius distributions measured for commercially available Nanogold (Red) and three independent thioglucose nanocrystal preparations (labeled with the month/year in which they were prepared). (C) Negative ion matrix assisted laser desorption/ionization mass spectrum of the thioglucose nanocrystal. The inset shows the m/z range centered at 15,500.

Mentions: As a potential heavy atom probe for a scattering interference ruler, we synthesized thioglucose passivated gold nanocrystals by the Brust method [10], [21]. In the presence of the radical scavenging agents Tris-HCl and ascorbic acid (Fig. 1A), the nanocrystal auto-scattering profile was stable for more than 200 seconds on the high-flux BESSRC-CAT 12ID-C beamline. We determined the size distribution of our nanocrystal preparation by decomposing the auto-scattering profile into a linear combination of basis profiles corresponding to hard sphere scatterers of varying diameter. The data indicate that the nanocrystals consist predominantly of 14 Å diameter spheres (Fig. 1B). The size distribution closely resembles the distribution we measure for commercially available Nanogold (Nanoprobes), and corresponds to a particle containing 75 gold atoms [22]. We also determined the molecular weight of the gold core of the particles using matrix assisted laser desorption/ionization mass spectrometry [23]. The dominant peak at m/z = 15,335 corresponds to 78 gold atoms and a sphere of 14 Å diameter (Fig. 1C). Thus the diameter of the nanocrystal probes is similar to the dimensions of an organic fluorophore.


A molecular ruler for measuring quantitative distance distributions.

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

Characterization of thioglucose passivated gold nanocrystals.(A) Five superimposed gold autoscattering frames measured in the presence of radical scavengers (top) are vertically offset from five autoscattering frames collected in the absence of radical scavengers (bottom). The frames were collected at one second intervals. Tris (70 mM) and ascorbic acid (10 mM) were used to suppress radiation damage. (B) Gold nanocrystal radius distributions measured for commercially available Nanogold (Red) and three independent thioglucose nanocrystal preparations (labeled with the month/year in which they were prepared). (C) Negative ion matrix assisted laser desorption/ionization mass spectrum of the thioglucose nanocrystal. The inset shows the m/z range centered at 15,500.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003229-g001: Characterization of thioglucose passivated gold nanocrystals.(A) Five superimposed gold autoscattering frames measured in the presence of radical scavengers (top) are vertically offset from five autoscattering frames collected in the absence of radical scavengers (bottom). The frames were collected at one second intervals. Tris (70 mM) and ascorbic acid (10 mM) were used to suppress radiation damage. (B) Gold nanocrystal radius distributions measured for commercially available Nanogold (Red) and three independent thioglucose nanocrystal preparations (labeled with the month/year in which they were prepared). (C) Negative ion matrix assisted laser desorption/ionization mass spectrum of the thioglucose nanocrystal. The inset shows the m/z range centered at 15,500.
Mentions: As a potential heavy atom probe for a scattering interference ruler, we synthesized thioglucose passivated gold nanocrystals by the Brust method [10], [21]. In the presence of the radical scavenging agents Tris-HCl and ascorbic acid (Fig. 1A), the nanocrystal auto-scattering profile was stable for more than 200 seconds on the high-flux BESSRC-CAT 12ID-C beamline. We determined the size distribution of our nanocrystal preparation by decomposing the auto-scattering profile into a linear combination of basis profiles corresponding to hard sphere scatterers of varying diameter. The data indicate that the nanocrystals consist predominantly of 14 Å diameter spheres (Fig. 1B). The size distribution closely resembles the distribution we measure for commercially available Nanogold (Nanoprobes), and corresponds to a particle containing 75 gold atoms [22]. We also determined the molecular weight of the gold core of the particles using matrix assisted laser desorption/ionization mass spectrometry [23]. The dominant peak at m/z = 15,335 corresponds to 78 gold atoms and a sphere of 14 Å diameter (Fig. 1C). Thus the diameter of the nanocrystal probes is similar to the dimensions of an organic fluorophore.

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