Developing new algorithms for single particle reconstruction of phase-plate cryo-EM images in the structural analysis of human Dicer (220kDa)

The phase-plate cryo-EM is a new imaging technique that is still under development. With a phase-plate apparatus inserted at the back-focal plane of a transmission electron microscope’s objective lens, this imaging technique can theoretically enhance the contrast of frozen-hydrated biological specimen with one order of magnitude than conventional cryo-EM, thus allowing the imaging of relatively small molecules.  A major hurdle of the application of phase-plate cryo-EM was the instability of the phase-plate and the lack of image processing algorithm for single particle analysis from the phase-plate EM dataset. We implemented a phase-plate specific filtering algorithm which allowed us to determine 20 Angstrom resolution structures of human Dicer and its complexes with different RNA substrates. This was the first proof of the capability of phase-plate cryo-EM in determining structure of relatively small macromolecules.

Taylor DW, Ma E, Shigematsu H, Cianfrocco MA, Noland CL, Nagayama K, Nogales E, Doudna JA*, and Wang HW*. (2013) Substrate-specific structural rearrangements of human Dicer. Nat. Struct. Mol. Biol., 20:662-670 (*co-corresponding authors)