DNA-origami scaffolds for structure determination .. (EM-FRAME)
DNA-origami scaffolds for structure determination by single particle analysis
Date du début: 1 mars 2013,
Date de fin: 28 févr. 2015
"Single-particle analysis of electron cryo-microscopy (cryo-EM) data can now provide near-atomic resolution information for large and high-symmetry protein complexes. However, application of the same method to small proteins is problematic, mainly because low signal-to-noise ratios hamper particle selection and orientation determination.The proposed research aims to tackle this problem by binding proteins to large 3D scaffolds made of DNA origami. The main idea is to use a hollow rectangular frame (called DNA alignment frame) with a dsDNA helix (called rotation axis) spanning the interior hole of the frame. By using a specific sequence, DNA-binding proteins may be bound to the rotation axis in a specific manner. The orientation of the bound protein may then be controlled by exploiting the helical character of the rotation axis in a series of experiments where the position of the binding site on the rotation axis is varied. Thus, experimental information about the orientation of the protein may be obtained, which will be used as probabilistic priors in a statistical 3D reconstruction approach. Application of this procedure to E. coli MutS and the human Trf2/Rap1 complex will serve as proof-of-principle and may lead to better understanding of their function."
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