Elucidating the Pol II-TCR-ELOF1-CNRL4CSA-E2-Ub Complex

We tried to solve the structure of the Pol II–TCR–ELOF1–C^NRL4^CSA–E2–Ub complex; however, we could only see the E2–Ub conjugate bound to the E3 ligase if we did not crosslink the sample and if we included a small amount of n-octyl glucoside before sample freezing. Thus, we prepared the Pol II–TCR–ELOF1–C^NRL4^CSA–E2–Ub complex in the same way as the Pol II–TCR–ELOF1– C^NRL4^CSA complex but with the addition of 10× excess of E2–Ub conjugate over Pol II and without sample crosslinking. After the dialysis step, we also supplemented the protein solution with 0.004% n-octyl glucoside. Under these conditions, the Pol II–TCR complex was falling apart during cryo-EM grid preparation, and Pol II exhibited very strong preferred orientation distribution. However, we could solve the structure of the C^NRL4^CSA–E2–Ub subcomplex from dissociated or not-bound ligase particles.