Yeast strains and plasmid constructs. The S. cerevisiae strains W303 (genotype: ade2-1, trp1-1, leu2-3,112, his3-11,15, ura3-1, can1-100), Noc4-TAP–Dhr1 K420A-Flag (genotype: NOC4-TAP::HIS3, pRS314 Dhr1 K420A-Flag, W303) (this study), Noc4-TAP–Dhr1-Flag4 and Dhr1-TAP–Dim1-Flag4 were used in this study. Genomic tagging was performed as described previously21. For plasmid construction, E. coli DH5a strain (Thermo Fisher Scientific) was used.
Split-tag tandem affinity-purification. Yeast strains used for split-tag tandem affinity purification, were harvested during the logarithmic growth phase. Frozen yeast cells were mechanically disrupted by a cryogenic grinding mill (Retsch MM400) in lysis buffer [60mM Tris-HCl pH 8.0, 40mM KCl, 50mM NaCl, 2mM MgCl2, 5% glycerol, 1mM DTT, 0.1% NP-40, EDTA-free protease inhibitors, 0.013 U/µl RiboLock RNase Inhibitors (Thermo Scientific)]. The lysate was cleared twice by centrifugation (10 min at 5000 rpm followed by 20 min at 17000 rpm, 4°C) and transferred to immunoglobulin G Sepharose 6 Fast Flow beads (GE Healthcare) for 3 hours at 4°C, followed by washing with buffer (60mM Tris-HCl pH 8.0, 40mM KCl, 15mM NaCl, 2mM MgCl2, 5% glycerol, 1mM DTT, 0.01% NP-40). Bound proteins were eluted from beads by TEV cleavage at 16°C for 45 min (buffer supplemented with a final concentration of 1U/ml RiboLock RNase inhibitors). For the second affinity purification step, the eluate was loaded onto Flag-agarose beads (Anti-Flag M2 Affinity Gel, Sigma–Aldrich) and incubated for 1 hour at 4°C. Beads were washed and eluted with buffer containing Flag peptide. The elution buffer for the cryo-EM analysis contained 60 mM Tris-HCl (pH 8.0), 50 mM NaCl, 5 mM MgCl2, 2% glycerol, 0.01% NP-40 and 1 mM DTT.
In vitro ATP assay. The final eluate of split-tag tandem affinity purifications was divided into two equal aliquots. Samples were supplemented with MgCl2 to a final concentration of 3mM and either AMP-PNP or ATP to a final concentration of 1mM. Samples were incubated at 10°C for 25 minutes and immediately loaded onto a sucrose gradient for separation of pre-ribosomes from released factors and RNAs.
Sucrose gradient ultracentrifugation. Eluates from the in vitro ATP assay were loaded onto a linear 10%–40% (w/v) sucrose gradient containing a buffer of 60 mM Tris-HCl (pH 8.0), 50 mM NaCl, 2 mM MgCl2, 0.003% NP-40 and 1 mM DTT, and centrifuged for 16 hours at 27,000 rpm at 4°C. The sucrose gradient was fractionated into 10 fractions and each fraction was split for RNA analysis (see below) or protein analysis. Protein samples were precipitated by 10% trichloroacetic acid (TCA), and TCA pellets resuspended in SDS sample buffer. Resuspended fractions were analyzed by 4%–12% gradient polyacrylamide gel electrophoresis (NuPAGE, Invitrogen), followed by staining with colloidal Coomassie Blue (Roti-Blue, Roth).
RNA extraction and Northern analysis. The RNA was isolated from the sucrose gradient fractions by phenol/chloroform and precipitated with ethanol as previously described4, 5. The extracted RNA was loaded onto an 8% polyacrylamide/8M urea gel to resolve smaller RNA species (U3 snoRNA) after denaturation with formaldehyde or loaded onto a 1.2% agarose gel to resolve larger pre-rRNA species (23S, 21S, and 20S) after denaturation with glyoxal. Followed by blotting to positively charged nylon membranes and UV crosslinking, the following 5'-end 32P-labeled DNA oligonucleotide probes were used for Northern analysis: 5'-GGTTATGGGACTCATCA-3' (probe for U3) and 5'-CGGTTTTAATTGTCCTA-3' (OMK002; probe for 23S, 21S, 20S).
Electron microscopy and image processing. Purified samples (3.5μl) were applied to pre-coated (2 nm) R3/3 holey-carbon-supported copper grids (Quantifoil), blotted for 2–3 s at 4°C, and plunge-frozen in liquid ethane using an FEI Vitrobot Mark IV. Data were collected on a Titan Krios cryo-electron microscope operating at 300 keV. All data were collected with a pixel size of 1.047 Å/pixel and within a defocus range of −0.8 to −2.5 μm using a K2 Summit direct electron detector under low-dose conditions, with a total dose of 44 e−/Å2. Original image stacks were dose-weighted, aligned, summed, and drift-corrected using MotionCor2 22. Contrast-transfer function (CTF) parameters and resolutions were estimated for each micrograph using CTFFIND4 and GCTF, respectively23, 24. Micrographs with an estimated resolution of less than 5 Å and an astigmatism of less than 5% were manually screened for contamination or carbon rupture.
In the end, a total of 6,218 good micrographs are selected for the Dhr1-Dim1 sample treated with ATP, while a total of 6,428 good micrographs for the control. Particle picking was carried out using Gautomatch without reference, resulting in 552,020 and 885,032 particles for the ATP treatment and control sample, respectively. Particle extraction was done in Relion 3.125, after that all the particles were imported into cryoSPARC26 for 2D classification and 3D classification until high resolution pre-40S particle with minimal orientation bias was reached, as shown in Supplementary Fig. 2. These particles were imported back to Relion 3.1 to do the final 3D classification (or focused 3D classification), CTF refinement, 3D refinement, Postprocessing and Local resolution filter.
Model building and refinement. The structure of the yeast primordial pre-40S ribosome (PDB IDs: 6ZQG)4 was used as initial references to generate the final models for the state Dis-D and Dis-E. In general, the model of state Dis-C was rigid body fitted using Coot27, followed by adjustment, such as removing the entire 40S Head part. Assembly factors that are not present in state Dis-D (such as, Utp14, Utp24, Rcl1, Dhr1 and U3 snoRNA et, al) and in state Dis-E (such as Bms1, Utp3/Sas10 and Mpp10) were manually removed afterward. The matured rRNA helix h27, and different conformation of the rRNA helices h1-h3 and h5 were de novo built in Coot27. The new immature rRNA helix h18 was built based on the shape of the density map and the secondary structure prediction.
The final models for the states Dis-D and Dis-E were real-space refined with secondary structure restraints using the PHENIX suite28. The final model evaluation was performed with MolProbity29. Maps and models were visualized, and figures were created with ChimeraX30.