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. 2019 May 22;8:e44904. doi: 10.7554/eLife.44904

Figure 1. Sequential steps in late cytoplasmic 60S subunit maturation.

(A) Cryo-EM reconstructions of six cytoplasmic maturation states (I–VI). States, overall resolution, changes in protein composition and rRNA conformation are indicated. The phosphatase (P) and zinc finger (Zn) domains of Yvh1 are indicated in state II. (B) Atomic models of pre-60S states I-VI with rRNA and biogenesis factors highlighted.

Figure 1.

Figure 1—figure supplement 1. Sample purification, cryo-EM image analysis and local resolution of pre-60S particles.

Figure 1—figure supplement 1.

(A) Schematic of pre-60S Lsg1-TAP affinity purification protocol. (B) Analysis of crude extract and supernatant by Coomassie-stained SDS-PAGE (left) and immunoblotting (right) with the indicated antibodies. (C) Representative micrograph from the Lsg1-TAP dataset. (D) Representative reference-free class averages of the Lsg1-TAP particles used for 3D classification. (E) Gold-standard Fourier shell correlation (FSC) curves after 3D refinement in RELION (Bai et al., 2013; Scheres, 2012b). (F) Surface views of unfiltered states I-VI coloured according to local resolution calculated in ResMap (Kucukelbir et al., 2014).
Figure 1—figure supplement 2. Cryo-EM data processing scheme.

Figure 1—figure supplement 2.

Maximum likelihood classification scheme and masks used to obtain native pre-60S maturation states I-VI. The two datasets were processed independently and identical classes from each dataset merged to yield six different states with five subclasses, highlighted in red at the bottom. Insets show the positions of the three masks (blue) on the 60S subunit (grey).
Figure 1—figure supplement 3. Cross-validation against overfitting.

Figure 1—figure supplement 3.

FSC curves are shown between the final refined atomic model of the states I-IV complexes and the reconstructions from all particles (orange); between the model refined in the reconstruction from only half of the particles and the reconstruction from that same half (red); and between that same model and the reconstruction from the other half of the particles (blue) calculated by REFMAC v5.8 (Amunts et al., 2014).
Figure 1—figure supplement 4. Local resolution for ribosome assembly factors, eL40 and uL16.

Figure 1—figure supplement 4.

Surface views of factors extracted from the indicated states are coloured by local resolution calculated in ResMap (Kucukelbir et al., 2014) and accompanied by atomic models. The Nmd3 N-terminus and Lsg1 are low-pass filtered, other factors unfiltered.
Figure 1—figure supplement 5. Protein-protein interaction network of pre-ribosomal particles purified by Lsg1-TAP.

Figure 1—figure supplement 5.

Biological replicates I (A) and II (B) of cross-linked pre-ribosomal particles immunopurified using Lsg1-TAP are shown. Proteins are represented as circles. Lysine residues are blue, crosslinks are black or grey (if multiple linkage sites were identified). Crosslinks are depicted at the residue level for Lsg1, Nmd3, Reh1 and Rei1. Inter-protein crosslinks are green; intra-protein crosslinks are purple.