6O6C

RNA polymerase II elongation complex arrested at a CPD lesion

6O6C の概要

• エントリーDOI: 10.2210/pdb6o6c/pdb
• EMDBエントリー: 0633
• 分子名称: DNA-directed RNA polymerase II subunit RPB1, DNA-directed RNA polymerases I, II, and III subunit RPABC4, RNA (5'-R(P*AP*UP*CP*GP*AP*GP*AP*GP*G)-3'), ... (15 entities in total)
• 機能のキーワード: rna polymerase, cpd, elongation complex, streptavidin grids, transcription, transferase-dna-rna complex, transferase/dna/rna
• 由来する生物種: Saccharomyces cerevisiae (Baker's yeast); 詳細
• タンパク質・核酸の鎖数: 13
• 化学式量合計: 486531.04

構造登録者

Lahiri, I.,Leshziner, A.E. (登録日: 2019-03-05, 公開日: 2019-06-26, 最終更新日: 2024-03-20)

主引用文献

Lahiri, I.,Xu, J.,Han, B.G.,Oh, J.,Wang, D.,DiMaio, F.,Leschziner, A.E.
3.1 angstrom structure of yeast RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion solved using streptavidin affinity grids.
J.Struct.Biol., 207:270-278, 2019

PubMed Abstract: 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 (for example, at its primary amines) 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, whether this method can be used for samples where interaction with the air-water interface has been shown to be problematic remains to be determined. Here we report a 3.1 Å structure of an 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 improved the angular distribution of its view, resulting in a high-resolution structure. This work shows that streptavidin affinity grids can be used to address known challenges posed by the interaction with the air-water interface.

PubMed: 31200019
DOI: 10.1016/j.jsb.2019.06.004

実験手法

ELECTRON MICROSCOPY (3.1 Å)