Diverse roles of assembly factors revealed by structures of late nuclear pre-60S ribosomes (original) (raw)

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Electron Microscopy Data Bank

Protein Data Bank

Data deposits

The cryo-EM density maps of state 1 and state 2 have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-6615 and EMD-6616, respectively. The atomic model of state 1 has been deposited in the Protein Data Bank (PDB) under accession number 3JCT. The XL–MS data have been deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org/) with the dataset identifier PXD003736.

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Acknowledgements

We thank the National Center for Protein Sciences (Beijing, China) for providing resource for cryo-EM data collection and computation. We also thank members of Woolford laboratory for reading the manuscript. This work was supported by the Ministry of Science and Technology of China (2013CB910404 to N.G. and 2014CB849800 to M.-Q.D.), the National Natural Science Foundation of China (31422016 and 31470722 to N.G., and 21375010 to M.-Q.D.) and National Institutes of Health grant R01GM028301 (to J.L.W.).

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Authors and Affiliations

  1. Ministry of Education Key Laboratory of Protein Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, 100084, Beijing, China
    Shan Wu, Kaige Yan, Yixiao Zhang, Yi Yuan, Zhifei Li, Chengying Ma, Jianlin Lei & Ning Gao
  2. Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, 15213, Pennsylvania, USA
    Beril Tutuncuoglu, Hailey Brown, Michael Gamalinda, Jelena Jakovljevic & John L. Woolford
  3. Graduate Program in Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100730, China
    Dan Tan & Meng-Qiu Dong
  4. National Institute of Biological Sciences, Beijing, 102206, China
    Dan Tan & Meng-Qiu Dong

Authors

  1. Shan Wu
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  2. Beril Tutuncuoglu
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  3. Kaige Yan
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  4. Hailey Brown
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  5. Yixiao Zhang
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  6. Dan Tan
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  7. Michael Gamalinda
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  8. Yi Yuan
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  9. Zhifei Li
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  10. Jelena Jakovljevic
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  11. Chengying Ma
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  12. Jianlin Lei
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  13. Meng-Qiu Dong
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  14. John L. Woolford
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  15. Ning Gao
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Contributions

N.G. and J.L.W. designed and directed experiments; B.K., H.B., M.G. and J.J. purified samples; D.T. and M.-Q.D. performed XL–MS; S.W. collected cryo-EM data (with J.L., Y.Y., Z.L., and C.M.), performed image processing (with Y.Z.), and analysed structures (with Y.K.). N.G., S.W. and K.Y. performed structural modelling. S.W., J.L.W. and N.G. wrote the paper.

Corresponding authors

Correspondence toJohn L. Woolford or Ning Gao.

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Extended data figures and tables

Extended Data Figure 1 Compositional analysis of Nsa1, Nog2 and Nmd3 particles.

a, Mostly non-overlapping assembly factors Nsa1, Nog2 and Nmd3 were used to purify sequential ribosome assembly intermediates. Proteins identified by mass spectrometry analysis were marked on the gel. Orange coloured proteins are only present in Nsa1-TAP particles, green coloured proteins are present both in Nsa1-TAP and in Nog2-TAP particles, light blue coloured proteins are present in all three purified particles to varying levels, dark blue coloured proteins are present only in Nog2-particles, pink coloured proteins are present both in Nog2- and Nmd3-particles in varying levels and yellow coloured proteins are present only in Nmd3-particles. TAP-tagged proteins are indicated by white asterisks. For gel source data, see Supplementary Fig. 1. b, The lifetimes of mostly non-overlapping ribosome assembly intermediates containing assembly factors Nsa1, Nog2 and Nmd3 are indicated. Assembly factors identified in each of Nsa1-TAP, Nog2-TAP and Nmd3-TAP associated samples were colour coded. The colour scheme is identical to that used in a. *Even though this protein was identified in all three intermediates, its levels decreased more than sevenfold from Nsa1-TAP particles to Nog2-TAP particles.

Extended Data Figure 2 Cryo-EM data processing of Nog2-particles.

a, Representative 2D class averages of Nog2-particles. b, A flow-chart for 3D classification of Nog2-particles (data batch 8–10, see Methods for details).

Extended Data Figure 3 Resolution estimation and model validation.

a, Representative micrograph of Nog2-particles. b, Local resolution map of the final density map of state 1. c, FSC curve for the final density map (state 1). The nominal resolution is 3.08 Å estimated using the gold-standard (FSC = 0.143) criterion. d, Atomic model cross-validation. Three FSC curves were calculated between the refined model (against Half1 map) and the final map (black), between the refined model with Half1 map (FSCwork, red), and between the refined model with Half2 map (FSCtest, blue) (see Methods for details).

Extended Data Figure 4 Local densities of representative regions for different assembly factors.

a–l, Cryo-EM densities of representative regions of assembly factors, superimposed with respective atomic models.

Extended Data Figure 5 Interaction network of Nog2 in the pre-60S particle.

a–g, Pairwise illustration of binding partners of Nog2 in the pre-60S particle. Residues of Nog2 involved in atomic contacts are coloured red with residue numbers labelled. H and L denote helix and loop, respectively. h, Interactions between rRNA components (H43, H68, H74, H75, H86, H92, H93) and Nog2. For clarification, H69 and H71 are not shown. The N terminus of Nog2 is located in a helical junction composed of H68, H74, H75 and H93.

Extended Data Figure 6 The NTD of Nog1 interacts with Nsa2 and Nog2.

a, Nsa2, Nog2 and Nog1 collectively stabilize H89 in a distinct conformation. Nog1 interacts with Nog2 and Nsa2 through its GTPase domain and NTD, respectively. b, The CTE of Nog1 interlocks with Rlp24 by wrapping around a long helix at the C-terminal end of Rlp24 (see also Fig. 3). c, d, Comparison of the CTE of Nog1 and the CTE of Rei1 in the polypeptide tunnel. Atomic models of state 1 (c) and 60S-Arx1–Alb1–Rei1 (d) (PDB accession number 5APN)16 are aligned using the 60S subunit. For clarification, only Arx1, Nog1 and Rei1 are shown. e, Superimposition of c and d. Four major places of steric clash between Rei1 and Nog1 are marked by asterisks.

Extended Data Figure 7 Mutual interactions between factors and r-proteins in the ITS2 subcomplex.

a, An overall view of the ITS2 subcomplex. b–d, L8 interacts with three factors: Nop15 (b), Cic1 (c) and Nop7 (d). e, L27 interacts with Nop53. f–h, L25 interacts with Rlp7 (f), Nop15 (g) and Nop53 (h). Residues involved in atomic interaction sites are labelled with sequence numbers. H, L, S denote helix, loop and strand of respective structures.

Extended Data Figure 8 Restructuring of rRNA helices in the central protuberance region by Nsa2, Rpf2, Rsa4, Rrs1 and Nog2.

a, Conformation of rRNA helices from the central protuberance (H80, H82-H88, 5S rRNA) in the pre-60S particle (state 1). b, Same as a, but for the mature 60S subunit. The mature 60S subunit was aligned to state 1 structure globally. c–g, Pairwise interactions between the central protuberance helices and factors are shown in separate panels.

Extended Data Figure 9 Structures of different assembly states of the pre-60S ribosomal particles.

a, Cryo-EM density maps of three premature states (1–3) and the mature state are displayed in transparent surface representation, superimposed with models of the 5S RNA, H38 and associated central-protuberance-binding factors. b, Zoom-in views of the central protuberance regions in a. For clarification, only atomic models are shown. Comparison of these four states indicates that the 5S RNP rotates to a near-mature state (state 2) after Rpf2–Rrs1 leave, and further release of Rsa4 in state 3 results in a ‘mature-like’ conformation for the 5S RNP. H38 from these four states is in a series of continuous changes coupled with the 5S RNP conformational maturation. c, d, Spatial relationship of the 5S RNP, H38, Rsa4 and Cgr1 in state 1 (c) and state 2 (d). Note that repositioning of H38 from state 1 to state 2 is coupled with a dramatic conformational change on the C-terminal end of Cgr1. e–h, Additional assembly factors identified in the density map of state 2. One piece of additional density between H38 and L1 contains a characteristic HEAT repeat, which contacts the L1 stalk in an inward position (e). The atomic model of Sda1 (PDB accession number 5FL8)14 fits well with the segmented density (f). For clarification, densities immediately above Sda1 are not shown in e and f. A large piece of additional density in the map of state 2, composed of the Rix1 subcomplex and Rea1 (g, h). The density assignment was facilitated by the cryo-EM structure of Rix1–Rea1 particles14. Superimposition of the atomic model of Rea1 (PDB accession number 5FL8)14 with the segmented density map of Rea1 (h).

Extended Data Table 1 Statistics of data collection, structural refinement and model validation

Full size table

Supplementary information

Supplementary Figure 1

This file contains the original gel image for Extended Data Fig. 1a. (PDF 171 kb)

Supplementary Table 1

This file shows cross-linked peptides identified in CL-MS data. (XLSX 36 kb)

Supplementary Table 2

This file shows the analysis results of CL-MS data. (XLS 1144 kb)

Overall structure of Nog2-particles in State 1

The cryo-EM density (sharpened) is first shown in surface representative, with densities of factors individually colored (as in Fig. 1). The rRNA and r-proteins are colored grey and beige, respectively. Groups of factors are then highlighted in zoom-in views. (MP4 29901 kb)

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Wu, S., Tutuncuoglu, B., Yan, K. et al. Diverse roles of assembly factors revealed by structures of late nuclear pre-60S ribosomes.Nature 534, 133–137 (2016). https://doi.org/10.1038/nature17942

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