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3.1Å structure of yeast RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion solved using streptavidin affinity grids

By Indrajit Lahiri, Jun Xu, Bong Gyoon Han, Juntaek Oh, Dong Wang, Frank Dimaio, Andres E Leschziner

Posted 22 Mar 2019
bioRxiv DOI: 10.1101/585547 (published DOI: 10.1016/j.jsb.2019.06.004)

Despite significant advances in all aspects of single particle cryo-electron microscopy (cryo-EM), specimen preparation still remains a challenge. During sample preparation, macromolecules interact with the air-water interface, which often leads to detrimental effects such as denaturation or adoption of preferred orientations, ultimately hindering structure determination. Randomly biotinylating the protein of interest and then tethering it to a cryo-EM grid coated with two- dimensional crystals of streptavidin (acting as an affinity surface) can prevent the protein from interacting with the air-water interface. Recently, this approach was successfully used to solve a high-resolution structure of a test sample, a bacterial ribosome. However, the general applicability of this method to samples where interaction with the air-water interface is problematic remains to be determined. Here we report a 3.1Å structure of a RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion (Pol II EC(CPD)) solved using streptavidin grids. Our previous attempt to solve this structure using conventional sample preparation methods resulted in a poor quality cryo-EM map due to Pol II EC(CPD)'s adopting a strong preferred orientation. Imaging the same sample on streptavidin grids led to a high-resolution structure with little anisotropy, showing that streptavidin affinity grids could be used as a general strategy to address challenges posed by interaction with the air-water interface.

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