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Cryo-EM structures of full-length Tetrahymena ribozyme at 3.1 Å resolution

Nature volume 596, pages 603–607 (2021)Cite this article

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Abstract

Single-particle cryogenic electron microscopy (cryo-EM) has become a standard technique for determining protein structures at atomic resolution1,2,3. However, cryo-EM studies of protein-free RNA are in their early days. The Tetrahymena thermophila group I self-splicing intron was the first ribozyme to be discovered and has been a prominent model system for the study of RNA catalysis and structure–function relationships4, but its full structure remains unknown. Here we report cryo-EM structures of the full-length Tetrahymena ribozyme in substrate-free and bound states at a resolution of 3.1 Å. Newly resolved peripheral regions form two coaxially stacked helices; these are interconnected by two kissing loop pseudoknots that wrap around the catalytic core and include two previously unforeseen (to our knowledge) tertiary interactions. The global architecture is nearly identical in both states; only the internal guide sequence and guanosine binding site undergo a large conformational change and a localized shift, respectively, upon binding of RNA substrates. These results provide a long-sought structural view of a paradigmatic RNA enzyme and signal a new era for the cryo-EM-based study of structure–function relationships in ribozymes.

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Fig. 1: Cryo-EM reconstruction of apo L-21 ScaI ribozyme.
Fig. 2: Structural insights in the peripheral regions.
Fig. 3: Cryo-EM structure of holo L-16 ScaI ribozyme reveals docked P1–P10 and conformational change of IGS.
Fig. 4: Localized shifts and mechanistic insights.

Data availability

The cryo-EM maps and associated atomic coordinate models of the apo L-21 and holo L-16 ScaI ribozymes have been deposited in the wwPDB OneDep System under EMD accession codes EMD-31385 and EMD-31386 and PDB ID codes 7EZ0 and 7EZ2, respectively.

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Acknowledgements

We thank R. N. Sengupta, D. Herschlag and I. Zheludev for discussions. Cryo-EM data were collected at SLAC and Stanford, and processed at SLAC, Stanford and Duyu High Performance Computing Center in Sichuan University. This work was supported by the National Institutes of Health (P41GM103832, R01GM079429, P01AI120943, and S10OD021600 to W.C.; R35GM112579 and R21AI145647 to R.D.); National Science Foundation (DGE-114747 to K.K. and DGE-1656518 to R.R.); Gabilan Stanford Graduate Fellowship to K.K.; Sichuan University start-up funding 20822041D4057 and Natural Science Foundation of China (NSFC82041016 and 32070049 to Z.S). We thank N. Lawless for manuscript editing.

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Author notes

  1. These authors contributed equally: Zhaoming Su, Kaiming Zhang, Kalli Kappel

Authors and Affiliations

  1. The State Key Laboratory of Biotherapy and Cancer Center, Department of Geriatrics and National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, China

    Zhaoming Su, Bingnan Luo & Yuquan Wei

  2. Department of Bioengineering and James H. Clark Center, Stanford University, Stanford, CA, USA

    Zhaoming Su, Kaiming Zhang, Shanshan Li, Grigore D. Pintilie & Wah Chiu

  3. MOE Key Laboratory for Membraneless Organelles and Cellular Dynamics, Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, China

    Kaiming Zhang & Shanshan Li

  4. Biophysics Program, Stanford University, Stanford, CA, USA

    Kalli Kappel, Michael Z. Palo, Ramya Rangan & Rhiju Das

  5. Department of Biochemistry and Department of Physics, Stanford University, Stanford, CA, USA

    Rhiju Das

  6. Division of CryoEM and Bioimaging, SSRL, SLAC National Accelerator Laboratory, Menlo Park, CA, USA

    Wah Chiu

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Contributions

Z.S., K.Z., K.K., R.D., W.C. conceived the project; K.K. and M.Z.P. prepared RNA samples; Z.S. and K.Z. collected cryo-EM data; Z.S., K.Z., S.L., and Y.W. processed cryo-EM data; Z.S., K.K., S.L., B.L. and G.D.P. built and refined atomic models; G.D.P, B.L., S.L. and R.R. validated the models and map–model correlation. All authors contributed to the preparation of the manuscript.

Corresponding authors

Correspondence to Zhaoming Su, Rhiju Das or Wah Chiu.

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The authors declare no competing interests.

Additional information

Peer review information Nature thanks Thomas Cech, Amedee des Georges and Andrej Lupták for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Cryo-EM single-particle reconstruction of the apo L-21 ScaI ribozyme.

Related to Fig. 1. a, Single-particle pipeline yields the final cryo-EM reconstruction with the corresponding angular distribution and local resolution map. The local resolution map shows more flexibility and lower resolution in the 4-nt bulge that connects stem P2.1 and P13, and towards the end of stem P6 and P9.2. b, FSC curve shows 3.1 Å resolution according to the 0.143 cutoff. c, Cryo-EM B factor64, which relates the number of particles to the map resolution attributed to cumulative experimental and computational factors that affect the final reconstruction.

Extended Data Fig. 2 Focused 3D classification of apo L-21 ScaI ribozyme reveals local conformational dynamics.

The regions of low local resolution, P9.2 (blue), P9.2–P9.1–P13 (orange) and P13 (green), were extracted. Focused 3D classification was performed and different classes were superimposed to show rotational and translational motions on P9.2 (left), P9.2–P9.1–P13 (middle) and P13 (right).

Extended Data Fig. 3 Q-score analyses of cryo-EM maps and models of both the apo L-21 and holo L-16 ScaI ribozymes.

Related to Fig. 1. a, Q-score analyses per residue of the apo L-21 (grey) and holo L-16 (blue) ScaI ribozyme cryo-EM models and maps. Black dashed line indicates average Q-score from nucleic acid cryo-EM models and maps at 3.1 Å resolution in the PDB. b, Cryo-EM model of apo L-21 ScaI ribozyme coloured according to Q-score per residue. Dashed boxes (black, blue and green) correspond to regions in the cryo-EM model with low Q-scores in a.

Extended Data Fig. 4 Detailed tertiary interactions in the core region of the apo L-21 ScaI ribozyme.

a–f, The P5–J5/5a–L9 region has a highly structured J5–5a junction in previous structures12,15. The cryo-EM structure shows tertiary interactions of C197 (a), C124 and A125 (b), G126, A324 and A325 (d, e) and G327 (f) in the minor groove of P5. g–k, Previous studies have shown that A183 and A184 in the A-rich bulge of the metal core, U259 and C260 from J6/J7 and U305 from J8/J7 are conserved and essential for catalytic site formation and splicing reactions65,66,67,68,69,70,71. The cryo-EM structure shows that U259 (h), C260 (i) and U305 (j, k) stack continuously and interact with P4 base triples. k, U168 from P5c stacks on the A-rich bulge and interacts with P4 in the minor groove, while pairing with G188 in P5a. l, The Hoogsteen base triple U277–A97–U300 is essential for substrate helix recognition72. m–q, In the P3–J3/4–P6–J7/3 region, the A-rich J3/4 and J7/3 were previously found to interact with P6 in the Azoarcus ribozyme17,73. Base triples formed by J3/4 were critical for catalysis65,70,71,74, and alterations in these regions result in RNA misfolding28,75. In the cryo-EM structure, A104 and A105 form A-minor interactions with P6, whereas A103 and A104 join A269 and A270 from J7/3 to form an adenosine cluster (m, n). The proposed A103–U271 reverse-Hoogsteen pair is not found; instead we observed a noncanonical A103–A270 pair74. p, q, The previously observed A-platform of A218–A219 is disrupted in the cryo-EM structure with P3 present12,29. A218 forms two A-minor interactions with C102–G272 and U273–U101 from P3, which also supports the conservation of this C–G pair in group Ib introns74. Black dashed lines indicate hydrogen bonds. The cryo-EM maps of all subpanels are visualized at 1σ threshold except for c, g, o (1.5σ).

Extended Data Fig. 5 Comparison between the previous 3.8 Å crystal structure of the mutated Tetrahymena ribozyme catalytic core (green) and the cryo-EM structure of the wild-type apo L-21 ScaI ribozyme construct (grey) shows minor differences.

Related to Extended Data Fig. 4. The overall r.m.s.d. for the catalytic core region (stem P3–P9) is 6.6 Å. a, The same view of the P5–J5/5a–P9 region as in Extended Data Fig. 4c. The nucleotide conformations generally agree well between two models; three mutations (U322C, U323G and U326A) are not involved in tertiary interactions. The r.m.s.d. for this region is 4.9 Å. b, The same view of the P4–P5a–J6/7–J8/7 region as in Extended Data Fig. 4g. In the crystal structure, U259A is slightly moved away from the G108–C213 base pair and disrupts this base triple interaction. A210G is moved far away from the wild-type position of A210, because there is no A46 in stem P2 to interact with in the crystal structure. The very top base quartet is much more compact in the cryo-EM structure compared to the crystal structure, probably owing to the presence of the peripheral domain that wraps around the catalytic core to make it more compact. The r.m.s.d. for this region is 5.7 Å. c, The same view of the P3–J3/4–P6–J7/3 region as in Extended Data Fig. 4o. The overall nucleotide conformations agree very well between the two models, except that A269G and A270 in the crystal structure are completely moved away and disrupt their interactions with A103, which is observed in the cryo-EM structure. The r.m.s.d. for this region is 1.7 Å. d, The same view as in Extended Data Fig. 4l. U277C disrupts the U277–A97–U300 base triple. The r.m.s.d. for this base triple is 2.7 Å. See also Supplementary Table 1.

Extended Data Fig. 6 Superposition of the apo L-21 ScaI ribozyme cryo-EM structure (grey) with previous crystal structures of the truncated and/or mutated Tetrahymena ribozyme, other group I introns and 5S rRNA loop E show global and local structural similarities.

a–d, Overlays of the cryo-EM structure (grey) with the Tetrahymena ribozyme P4–P6 Delta C209 (a; blue, PDB 1HR2); the mutated Tetrahymena ribozyme P3–P9 (b; green, PDB 1X8W); the Azoarcus ribozyme (c; violet, PDB 1U6B) and the phage Twort ribozyme (d; yellow, PDB 1Y0Q). e, P5c region of the wild type P4–P6 crystal structure (blue, PDB 1GID). f, 5S rRNA loop E crystal structure (red, PDB 354D).

Extended Data Fig. 7 Cryo-EM single-particle reconstruction of the holo L-16 ScaI ribozyme.

Related to Fig. 3. a, Single-particle pipeline yields the final cryo-EM reconstruction with the corresponding angular distribution and local resolution map. The local resolution map shows more flexibility and lower resolution in the 4-nt bulge that connects stem P2.1 and P13, and towards the end of stem P6 and P9.2. b, FSC curve shows 3.1 Å resolution according to the 0.143 cutoff. c, Cryo-EM B factor.

Extended Data Fig. 8 Comparisons of apo L-21 and holo L-16 ScaI ribozyme cryo-EM models with previous crystal structures show structural conservation and metal ion shifts in the guanosine binding site among group I introns.

Related to Fig. 4. a–c, The apo L-21 ScaI ribozyme adopts a preorganized guanosine binding site (grey) that superimposes with previous crystal structures of mutated P3–P9 of the Tetrahymena ribozyme (a; green, PDB 1X8W), the Azoarcus ribozyme (b; violet, PDB 1U6B), and the phage Twort ribozyme (c; yellow, PDB 1y0q). d–g, The holo L-16 ScaI ribozyme (sky blue) superimposes with apo L-21 ScaI ribozyme (d; grey), the Azoarcus ribozyme (e; violet), the phage Twort ribozyme (f; yellow), and mutated P3–P9 of the Tetrahymena ribozyme (g; green). MC is absent in the apo L-21 ScaI ribozyme, whereas MC in the Azoarcus ribozyme is shifted compared to the holo L-16 ScaI ribozyme. Dash line indicates metal ion coordination with surrounding atoms.

Extended Data Fig. 9 Metal ion validations by distance and Q-score analysis, and illustrations of the apo L-21 and holo L-16 ScaI ribozyme cryo-EM structures compared with previous crystal structures.

a, Distances between metal ions and other atoms in the apo L-21 ScaI ribozyme model. b, Distances between metal ions and other atoms in the holo L-16 ScaI ribozyme model. c, Q-score analysis per metal ion of the apo L-21 and holo L-16 ScaI ribozyme cryo-EM models and maps. d, Metal core region of the holo L-16 ScaI ribozyme, visualized at 1.1σ threshold. e, Comparisons of the apo L-21 (grey) and holo L-16 (sky blue) ScaI ribozyme cryo-EM models with P4–P6 Delta C209 (blue, PDB 1HR2) and mutated P3–P9 of the Tetrahymena ribozyme (green, PDB 1X8W) in the same view as d. f, Catalytic site of the holo L-16 ScaI ribozyme, visualized at 1.4σ threshold. g, Comparisons of the apo L-21 (grey) and holo L-16 (sky blue) ScaI ribozyme cryo-EM models with the Azoarcus ribozyme (violet, PDB 1U6B), mutated P3–P9 of the Tetrahymena ribozyme (green, PDB 1X8W), and the phage Twort ribozyme (yellow, PDB 1Y0Q) in the same view as f. See also Supplementary Table 2.

Extended Data Table 1 Cryo-EM data collection, processing, and model refinement statistics of the apo L-21 and holo L-16 ScaI ribozymes
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Video 1

Cryo-EM map and model of the apo L-21 ScaI Tetrahymena ribozyme with zoom in views of newly discovered tertiary interactions and representative metal ions shown in Figure 2, accompanied with the updated secondary structure.

Video 2

Comparison of the apo L-21 and holo L-16 Tetrahymena ribozyme cryo-EM maps and models reveals substantial conformational change upon substrate binding.

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Su, Z., Zhang, K., Kappel, K. et al. Cryo-EM structures of full-length Tetrahymena ribozyme at 3.1 Å resolution. Nature 596, 603–607 (2021). https://doi.org/10.1038/s41586-021-03803-w

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