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A simple method for tethering microbeads using single molecules of DNA is explained. We describe how to use video microscopy and particle tracking to measure the trajectories of the microbeads' motion. The trajectories are analyzed and compared to different models of tethered particle motion. In addition, the data are used to measure the elasticity of the DNA (its spring constant), and the DNA persistence length. V

SARS was epidemic in 2003 worldwide. SARS-CoV helicase plays critical roles in viral replication, and has been proposed to be a potential candidate for anti-SARS therapy. We use single molecule fluorescence resonance energy transfer to examine the unwinding and rewinding mechanism of nsP13 helicase on partial DNA duplexes as a function of protein, ATP concentration, and tail length. Our results reveal that the tail length of the substrates determines the total amount of DNA unwound by increasing the number of proteins loaded. In contrast, unwinding rate and step size increase as a function of the protein and ATP concentration for the partial duplex with a long tail (45nts long), but independent of protein concentration for the short tail (30nts long). We also observed a repetitive unwinding displaying multiple rounds of reunwinding and re-zipping events where re-unwinding becomes favorable at higher protein concentration. We also found that the relative extent of constitutive unwinding and repetitive fluctuation is defined by the modality of DNA-Protein complex in the presence or absence of ATP concentration. The ratio between them determines the processivity of the cooperative helicases in tandem. In general, our results identify the important cellular parameters, governing the cooperative unwinding and repetitive rewinding behavior of helicase. This is a new attempt to understand the complicate behavior of unwinding motor cohorts at the single molecule resolution.

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set solutions, final presentations, peer feedback, pre-and post course surveys, as well as a comprehensive structure solution set to be solved in a longterm post-test. Participants showed good problem solving-and very good crystallographic communication and feedback skills. Their confidence level applying x-ray crystallography techniques increased during the course. Most importantly, three months after the course, >80% of participants were able to solve and refine unknown diffraction datasets to publication quality level.