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. 2017 Dec 14;171(7):1599–1610.e14. doi: 10.1016/j.cell.2017.11.039

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© 2017 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Figure S1 — Affinity Purification of Nucleolar Pre-60S Particles for Cryo-EM Analysis, Related to Figure 1

(A and B) Split affinity purifications of pre-60S intermediates (upper panels) purified through Nsa1-TAP Flag-Ytm1 (A) and Rix1-TAP Rpf2-Flag (B). Final eluates were used for cryo-EM and analyzed by SDS-PAGE and Coomassie staining. Major protein bands were identified by mass-spectrometry and are labeled on the right side of the gels. The bait proteins are shown in bold and indicated by asterisks. The Rix1-TAP Rpf2-Flag purification purifies nucleolar (state E) and nucleoplasmic (state F) pre-60S particles (B). Nucleoplasmic assembly factors are indicated in green and the Rix1 subcomplex members (Rix1, Ipi3, Ipi1) in blue. Growth analysis (lower panels) of the Nsa1-TAP Flag-Ytm1 (A) and the Rix1-TAP Rpf2-Flag (B) strains in comparison to the untagged wild-type strain (DS1-2b). Cells were spotted in 10-fold serial dilution on YPD medium and cell growth at the indicated temperatures was monitored after 2 days.

(C) Affinity purifications of the indicated plasmid-encoded Ytm1 variants fused to an N-terminal TAP-Flag tag and expressed from the endogenous promoter. The plasmids were transformed into a wild-type strain (W303). Final eluates of the purifications were analyzed by SDS-PAGE and Coomassie staining and co-purifying proteins are indicated on the right side of the gel and bands corresponding to the Ytm1 bait proteins are marked with an asterisk. Proteins decreased in the Ytm1 ΔUBL/E80A purifications compared to the Ytm1 wild-type particle are shown in red and factors increased on the mutant particles are indicated in green.

(D) Affinity purification of the Ytm1 E80A particle used for cryo-EM analyzed by SDS-PAGE. Proteins bands were identified by mass-spectrometry and are labeled on the right side of the Coomassie stained gel.