A molecular ruler for measuring quantitative distance distributions.
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
Show MeSH
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. Related in: MedlinePlus |
Related In:
Results -
Collection
getmorefigures.php?uid=PMC2566812&req=5
Mentions: Finally, we investigated the length resolution of the scattering interference ruler as well as its ability to recover complex distributions. First, we measured the end-to-end distance distributions of four DNA duplexes with 10, 11, 12 and 13 base pairs. The distributions overlap, but consistently tend to longer distances (Fig. 6A). Thus, the ruler is capable of resolving single base-pair increments in DNA length, corresponding to approximately one-fifth of the diameter of the gold nanocrystal. Next, we created a sample of heterogeneous length by mixing equal quantities of labeled 10 and 25 base-pair duplexes. The corresponding distribution shows two peaks separated by 45 Å, in relative proportions of 45% and 55% (Fig. 6B). Third, we measured the end-to-end distribution of a floppy macromolecule, a nicked 27 base-pair duplex consisting of two 12 base-pair duplexes linked by three unpaired T nucleotides [31]. At 1 M NaCl, the nicked duplex exhibits a broad, unimodal distribution (Fig. 6C), with a mean distance of 93 Å. The roughly triangular shape of the distribution is expected for a pair of weakly-interacting freely-jointed segments. This type of measurement should provide a powerful test of theoretical models for the conformational fluctuations of macromolecules. Taken together, the results establish that the scattering interference ruler is applicable to heterogeneous samples that exhibit a wide range of different probe-probe separation distances. |
View Article: PubMed Central - PubMed
Affiliation: Department of Biochemistry, Stanford University, Stanford, California, USA.