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

RNA preparation. L-21 ScaI ribozyme was prepared as previously described 10 . Briefly, the DNA template was amplified from the pT7L-21 plasmid 63 , then RNA was prepared through in vitro transcription in a reaction containing 0.2 μM DNA template, 40 mM Tris·HCl, pH 8.1, 25 mM MgCl 2 , 3.5 mM spermidine, 0.01% TritonX-100, 40 mM DTT, 4% PEG 8000, 3 mM NTPs, and 7.5 U/μL T7 RNA polymerase (New England Biolabs). The transcription reaction was incubated at 37°C for 1 hour. The RNA was then isolated by ethanol precipitation, then purified on an 8% 29:1 acrylamide:bis, 7 M urea polyacrylamide gel. The gel was allowed to set overnight, then the precipitated RNA was mixed with loading buffer containing 95% formamide, 10mM EDTA, 0.1% xylene cyanol, and 0.1% bromophenol blue, and loaded on the gel. The gel was run at 25 W for 2 hours, then visualized briefly with a 254-nm UV lamp, held far from the gel to minimize RNA damage 64 . RNA was eluted from the gel overnight in RNase-free water at 4°C, then purified with Zymo RNA Clean and Concentrator columns (Zymo Research). The DNA template of L-16 ScaI ribozyme was amplified from the pT7L-21 plasmid using forward primer 5′-TTCTAATACGACTCACTATAGGTTTGGAGGGAAAAGTTATCAGGCATGCACCTGGTAGC-3′ and reverse primer 5′-ACTCCAAAACTAATCAATATACTTTCGCATACAAATTAGTTCCCAGCGGCTCC-3′. RNA was prepared using the TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific) according to the manufacturer’s protocol, then purified with RNA Clean and Concentrator-25 columns (Zymo Research) and PAGE purified as described above for L-21 ScaI ribozyme. RNA was eluted from the gel using the ZR small-RNA PAGE Recovery Kit (Zymo Research) and then ethanol precipitated. RNA oligonucleotide substrate S1 (5′-UCG*UAACC) and S2 (5′-CCCUCU), where * indicates a phosphorothioate bond, were acquired from Integrated DNA Technologies. A phosphorothioate-substituted substrate was selected to capture the complex mimicking the second step of splicing based on previous work showing that a similar ligation reaction catalyzed by Tetrahymena ribozyme was significantly inhibited by substitution of phosphorothioate in the R p isomeric form at the scissile phosphate (less than 5% product after 3 hours at 10 μM ribozyme concentration) 65 . Similarly, phosphorothioate substitution in the equivalent substrate for catalysis of the second step of splicing by the Azoarcus ribozyme reduced the reaction rate by a factor of >10 5 and ~14 for the R p and S p isomeric forms, respectively 39 . Cryo-EM sample preparation. To prepare L-21 and L-16 ScaI ribozyme samples for cryo-EM analysis, RNAs (20 μM or 15 μM final concentration, respectively) were denatured at 90°C for 3 min in 50 mM Na-HEPES, pH 8 and cooled to room temperature for 10 min. MgCl 2 was added to a final concentration of 10 mM and the samples were incubated at 50°C for 30 min. Ribozyme samples were again cooled to room temperature for 10 min. At this point, L-21 ScaI ribozyme was kept on ice, while substrates S1 and S2 (75 μM final concentration each) were added to L-16 ScaI ribozyme and the sample was incubated at room temperature for 20 min to form the holoenzyme complex before being placed on ice. A total of 3 μl of the Tetrahymena ribozyme sample was applied onto glow-discharged (30 s) 200-mesh R2/1 Quantifoil Cu grids. The grids were blotted for 3 s in 100% humidity with no blotting offset and rapidly frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher). Cryo-EM single particle data acquisition and data processing. The frozen grids of apo L-21 ScaI ribozyme were loaded in Titan Krios (Thermo Fisher) operated at 300 kV, condenser lens aperture 50 μm, spot size 7, parallel beam with illuminated area of 0.85 μm in diameter. Microscope magnification was at 215,000× (corresponding to a calibrated sampling of 0.65 Å per physical pixel). Movie stacks were collected automatically using EPU software on a K2 direct electron camera equipped with a Bioquantum energy filter with an energy slit of 20 eV (Gatan), operating in counting mode at a recording rate of 5 raw frames per second and a total exposure time of 5 seconds, yielding 25 frames per stack, and a total dose of 75 e − /Å 2 . A total of 7,577 movie stacks were collected with defocus values ranging between −0.3 and −1.5 μm. These movie stacks were motion corrected using Motioncor2 66 . After CTF correction by CTFFIND4 67 , 7,469 micrographs were subjected to EMAN2.2 for neural network particle picking 68 . A total of 1,658,961 particles were extracted in Relion3 69 with the box size of 320 pixels. After two rounds of 2D classifications, the best classes by visual examination were subjected to EMAN2.2 to build the initial model, and a total of 1,559,933 particles were subjected to 3D classification in Relion3. The major class showing RNA features, including 415,918 particles, were subjected to auto refinement. The initial auto refinement result was subjected to Bayesian polishing followed by another round of auto refinement 70 . A sharpening B-factor of −14 Å 2 was applied to the resulting cryo-EM map to yield the final sharpened map at 3.1 Å global resolution estimated by the 0.143 criterion of FSC curve. Frozen grids of the holo L-16 ScaI ribozyme were loaded in Titan Krios (Thermo Fisher) operated at 300 kV, condenser lens aperture 70 μm, spot size 5, parallel beam with illuminated area of 1.1 μm in diameter. Microscope magnification was at 105,000× (corresponding to a calibrated sampling of 0.86 Å per physical pixel). Movie stacks were collected automatically using EPU software on a K3 direct electron camera equipped with a Bioquantum energy filter with an energy slit of 15 eV (Gatan), operating in counting mode and a total exposure time of 2.5 seconds, yielding 30 frames per stack with a total dose of 50 e − /Å 2 . A total of 5,559 movie stacks were collected with defocus values ranging between −0.8 and −2.0 μm. The data was processed using the above-mentioned protocol, and a total of 230,386 particles were subjected to auto refinement, Bayesian polishing and postprocessing with a sharpening B-factor of −30 Å 2 to yield the final sharpened map at 3.1 Å global resolution estimated by the 0.143 criterion of FSC curve. Both local resolution maps were determined in Relion3 and the final map was low-pass filtered accordingly and displayed in UCSF Chimera 71 . Cryo-EM model building and refinement. The initial Tetrahymena ribozyme models of both apo and holo cryo-EM structures were built with DRRAFTER 72 , then manually adjusted and rebuilt with Coot as needed 73 . The models were refined with Phenix.real_space_refine 74 , yielding an averaged model–map correlation coefficient (CCmask) of 0.79 and 0.82, respectively. The final model was validated by MolProbity 75 and Q-score analysis 33 . Secondary structure diagrams were prepared with RiboDraw aided by manual adjustment ( https://github.com/ribokit/RiboDraw ). Metal ion identification and validation in cryo-EM models. Additional densities in our cryo-EM maps after fitting the RNA models are modeled as metal ions. These densities are also observed in both half maps of the reconstruction, which is another means of validating these metal ions. Several metal ions in the cryo-EM structures validate previous biochemical results: M 4 and M 5 have been previously identified to adopt inner-sphere coordination by metal-ion rescue experiments 76 ; residues that make inner-sphere contacts with M 2 , M 15 , M 16 , M 18 and M 19 have been suggested as metal binding sites in a phosphorothioate interference assay 77 , 78 .

Supplementary Material 1728251_Sup_tab1 1728251_Sup_tab2 1728251_Sup_Vdo1 1728251_Sup_Vdo2