www.sciencemag.org/cgi/content/full/science.aag2235/DC1 Supplementary Materials for Structure of a yeast catalytic step I spliceosome at 3.4 Å resolution Ruixue Wan, Chuangye Yan, Rui Bai, Gaoxingyu Huang, Yigong Shi* *Corresponding author. Email: shi-lab@tsinghua.edu.cn Published 21 July 2016 on Science First Release DOI: 10.1126/science.aag2235 This PDF file includes: Materials and Methods Figs. S1 to S14 Tables S1 to S4 Full Reference List Wan & Yan et al Materials and Methods Purification of the spliceosomal complexes Purification of the spliceosomal complexes from the yeast Saccharomyces cerevisiae (S. cerevisiae) was as described (42). EM data acquisition and processing Preparation of the cryo-electron microscopy (cryo-EM) sample and acquisition of the EM micrographs were as described (42). After the manual check procedure, a data set of 761,767 particles was produced for further processing (42). Image processing The overall shape and appearance of the previously characterized spliceosomal complexes (26, 30) served as a useful guide for our visual judgment of which 3D class averages may correspond to the Bact, C, and ILS complexes. Our preliminary analysis suggested that the spliceosomal C and ILS complexes may be represented by about 34.5 percent of the total particles (42). The same strategy of 3D classification as that used for the reconstruction of the Bact spliceosome (42) was applied to the spliceosomal C complex. To avoid the problem of discarding good particles, we simultaneously performed three independent 3D classifications (K=5, 6, and 7) (Fig. S1). Then we merged all seven classes that appear to represent the C complex; using an in-house script, we removed the duplicated particles according to the unique index 2 Wan & Yan et al of each particle given by RELION (69). This procedure resulted in the selection of 442,876 particles, representing 58.1 percent of the total. The same procedure was repeated one more time, yielding 161,066 good particles (21.1 percent of the total). Following auto-refinement, these 161,066 particles gave a reconstruction with an average resolution of 3.95 Å. After per-particle motion correction and radiationdamage weighting (known as particle polishing) (70), these polished particles give a reconstruction with an improved average resolution of 3.41 Å following autorefinement (Figs. S1 & S2). The core regions of the C complex display excellent EM density map; but some of the peripheral regions only have continuous density after the maps were low-pass filtered to 10 Å (Fig. S2D). To improve the density at the peripheral regions, we performed a third round 3D classification (K=5) without alignment using the refined polished particles. 80,367 particles in one major class, representing 49.9 percent of the total input, gave an average resolution of 3.65 Å (Figs. S3 & S4). Although the resolution is a bit lower, the quality of the EM density maps in some regions is better than that of the 3.41 Å reconstruction. Finally, an additional round of 3D classification was performed, and one class with 20,686 particles was identified and gave an average resolution of 4.6 Å. This map gave a better density for the U2 snRNP region (Fig. S3). The angular distribution of the particles used for the reconstruction of the C complex at 3.65 Å resolution is reasonable (Fig. S4A), and the refinement of the atomic coordinates did not suffer from severe overfitting (Fig. S4B). The resulting density maps show clear 3 Wan & Yan et al features for the secondary structural elements and amino acid side chains for most protein components of the C complex (Figs. S5-S7). The RNA elements and their interacting proteins are also well defined by the EM density maps (Fig. S8-S11). Reported resolutions are calculated on the basis of the gold-standard FSC 0.143 criterion (Fig. S4C), and the FSC curves were corrected for the effects of a soft mask on the FSC curve using high-resolution noise substitution (71). Prior to visualization, all density maps were corrected for the modulation transfer function (MTF) of the detector, and then sharpened by applying a negative B-factor that was estimated using automated procedures (72). Local resolution variations were estimated using ResMap (73). Model Building and refinement Due to a wide range of resolution limits for the various regions of the spliceosomal C complex, we combined de novo model building and homologous structure modeling to generate an atomic model (Tables S1-S4). Identification and docking of the components of the C complex were facilitated by the structures of the ILS complex at 3.6 Å resolution (10, 40) and the Bact complex at 3.5 Å resolution (42). The protein components derived from the associated PDB accession code (3JB9 for the ILS complex from S. pombe) are summarized in Table S2. These structures were docked into the density map using COOT (74) and fitted into density using CHIMERA (67). 4 Wan & Yan et al The structure of the spliceosomal Bact complex (42) greatly facilitates the atomic modeling of 13 protein components. The atomic coordinates of Prp8, Snu114, Clf1, Cef1, Bud31, Cwc2, Cwc15, Ecm2, Prp45, Prp46, Cwc21, Cwc22, and Prp17 from the cryo-EM structure of the Bact complex (42) were individually docked into the density maps and manually rebuilt using COOT (74). For each of the remaining four protein components that lacks a homologous structure, de novo model building was performed, annotated with the label “De novo building” in Table S2. These protein components include the NTC proteins Syf2 and Isy1, and the splicing factors Yju2 and Cwc25. The chemical properties of proteins and amino acids were considered to facilitate model building. Sequence assignment was guided mainly by bulky residues such as Phe, Tyr, Trp and Arg. Unique patterns of sequences were exploited for validation of residue assignment. The RNA sequence assignment was greatly aided by the structure of the spliceosomal Bact complex (42). The RNA sequences were manually built using COOT (74). The RNA nucleotides, together with all protein components, were refined using REFMAC in reciprocal space (75). To further improve the geometries of the RNA nucleotides, the RNA elements alone were adjusted using RCrane (76). The conformations of the RNA components were further refined using phenix.erraser (77). ERRASER is a Rosetta program for modeling RNA nucleotides into density. 5 Wan & Yan et al On the basis of the EM density maps, we identified five metal ions that are bound by nucleotides in the ISL of U6 snRNA. On the basis of previous biochemical characterizations (1, 5, 8, 9), these metal ions were tentatively assigned as Mg2+. In all cases, the local maxima of the EM density that may correspond to ions are 2.0–2.4 Å away from the oxygen atoms of the phosphate groups, consistent with the metrics for Mg2+ coordination (78-82). In contrast, K+ is usually measured at 2.8–3.5 Å from the coordinating ligands (78, 79, 83). Therefore, the densities seen here are likely those of Mg2+. Despite the high likelihood, we acknowledge that at the reported resolution we cannot unambiguously assign these metal ions to Mg2+. In addition, we cannot conclusively differentiate Mg2+ from water molecules, although water molecules should be much less visible in the EM density maps. Structure refinement of the individual protein was carried out using phenix.real_space_refine application in PHENIX in real space (84) with secondary structure and geometry restraints to prevent over-fitting. The final overall model was refined against the overall 3.65 Å map using REFMAC in reciprocal space (75), using secondary structure restraints that were generated by ProSMART (85). Overfitting of the overall model was monitored by refining the model in one of the two independent maps from the gold-standard refinement approach, and testing the refined model against the other map (86) (Fig. S4B). Protein structures in the C complex were individually validated through 6 Wan & Yan et al examination of their Molprobity scores, statistics of Ramachandran plots, and EMRinger scores (Table S3). Only protein structures that were solved by homology modeling or de novo building in Table S2 are included for this practice. For obvious reasons, those structures that were fitted into the cryo-EM density maps by rigid-body docking were omitted for such model validation. Molprobity scores were calculated as described (87) . EMRinger scores were calculated as described (88). EMRinger is a side chain–directed model and map validation tool for cryo-EM structure determination. EMRinger evaluates how precise an atomic model is fitted into the cryo-EM map during refinement. EMRinger scores should be above 1.0 for wellrefined structures with maps in the 3- to 4-Å range. The RNA nucleotides in the C complex were validated directly by the Molprobity server, and the results are shown in Table S4. 7 Wan & Yan et al Table S1. Cryo-EM data collection and refinement statistics. Data collection EM equipment Voltage (kV) Detector Pixel size (Å) Electron dose (e-/Å2) Defocus range (µm) Reconstruction Software Number of used Particles Accuracy of rotation (˚) Accuracy of translation (pixels) Final Resolution (Å) Model building software Refinement Software Map sharpening B-factor (Å2) Average Fourier shell correlation R-factor Model composition Protein residues RNA nucleotides GTP Validation R.m.s deviations Bonds length (Å) Bonds Angle (˚) Ramachandran plot statistics (%) Preferred Allowed Outlier FEI Titan Krios 300 Gatan K2 1.306 45.6 1.6~2.6 RELION 1.4 161,066 / 80,367 0.441 / 0.494 0.402 / 0.405 3.41 / 3.65 Coot & Rosetta Phenix & Refmac -83.4 0.893 0.245 8,587 434 1 0.010 1.343 92.48 5.94 1.59 8 Wan & Yan et al Table S2 Summary of model building for the yeast spliceosomal C complex. U5 snRNP U6 snRNP U2 snRNP NTC/Prp19 Complex NTC-Related proteins Known Splicing Factors Step2 proteins Pre-mRNA Molecule U5 snRNA Length 214 Prp8 2413 Snu114 SmB1 SmD1 SmD2 SmD3 SmE1 SmF1 SmG1 U6 snRNA U2 RNA 984 196 146 110 101 94 86 77 112 nt 1175 nt Lea1 Msl1 SmB1 SmD1 SmD2 SmD3 SmE1 SmF1 SmG1 Clf1 238 111 196 146 110 101 94 86 77 687 Domain/Region 28:183 N-terminal Domain (127:839) RT finger/palm (840:1253) Thumb/X (1254:1377) Linker (1378:1650) Endonuclease (1651:1829) RNaseH-like (1830:2085) Jab1/MPN (2148-2398) 67:975 Sm fold Sm fold Sm fold Sm fold Sm fold Sm fold Sm fold 1:106 1:48 53:107 109:191 LRR domain RRM domain Sm fold Sm fold Sm fold Sm fold Sm fold Sm fold Sm fold TPR domain (40:275) - PDB code From Bact Modeling Homology modeling Resolution (Å) 2.9~4.0 From Bact Homology modeling 2.9~4.0 From Bact Not modeled Homology modeling ~4.0 2.9~4.0 From Bact Rigid docking From Bact From Bact 3JB9 3JB9 3JB9 Homology modeling Homology modeling RNA duplex Rigid Docking Rigid Docking Rigid Docking 3JB9 Rigid docking From Bact 3JB9 3JB9 Homology modeling Rigid docking Rigid docking 3.0~4.5 ~30 ~30 From Bact 3JB9 3JB9 3JB9 Homology modeling Homology modeling Rigid docking Rigid docking 3.0~3.5 3.0~3.5 ~30 ~30 4.0~6.0 2.9~4.0 2.9~4.0 5.0~8.0 5.0~8.0 5.0~8.0 5.0~8.0 5.0~8.0 Syf1 859 Cef1 590 Prp19 503 Myb Domain (9:111) 145:253 - Syf2 215 92:211 - De novo building 3.5~4.0 Isy1 235 2:96 - De novo building 3.5~4.0 Snt309 175 - 3JB9 Rigid docking ~30 Bud31 157 1:157 From Bact Homology modeling 3.0~3.5 Cwc2 339 1:261 Homology modeling 3.0~4.0 Cwc15 175 3:41/127:175 Homology modeling 3.0~4.0 Ecm2 364 RRM domain (3:288) Homology modeling 3.0~5.0 Prp45 379 34:247 Homology modeling 3.0~4.0 Prp46 451 WD40 domain (111:447) Homology modeling 2.9~3.5 Cwc21 Cwc22 135 577 From Bact From Bact Cwc25 Yju2 Prp17 179 278 455 2:28 MIF4G domain (11:263) MA3 domain (279:485) N-terminal domain (2:42) 2:116 50:75 3JB9 Homology modeling Rigid docking Homology modeling De novo building De novo building Homology modeling 3.0~4.0 4~10 3.0~3.5 2.8~3.2 3.0~4.0 3.0~4.2 Pre-mRNA - 57nt From Bact De novo building 2.9~4.5 9 Wan & Yan et al Table S3 Summary of model validation for individual proteins of the yeast C complex (Proteins solved by homology modeling or de novo building in Table S2 are included here). *EMRinger: side chain–directed model and map validation tool for 3D cryo-electron microscopy Molecule Molprobity Scores Ramachandran plot statistics (%) EMRinger* Score Preferred Allowed Outlier Prp8 2.16 3.13 92.44 6.04 1.52 Snu114 2.19 2.97 91.86 6.88 1.26 Clf1 2.24 2.57 93.28 6.72 0.00 Syf2 1.71 96.94 2.04 1.02 1.70 Isy1 2.35 86.02 12.90 1.08 1.65 Cef1 2.10 93.27 4.33 2.40 2.54 Bud31 2.05 90.32 8.39 1.29 1.72 Cwc2 2.09 91.44 7.39 1.17 2.71 Cwc15 2.02 87.30 12.70 0.00 3.99 Ecm2 2.32 87.01 11.86 1.13 1.59 Prp45 1.77 91.19 6.74 2.07 2.40 Prp46 2.33 88.96 8.96 2.09 3.01 Cwc22 2.00 92.68 5.37 1.95 3.19 Cwc25 1.75 94.87 2.56 2.56 3.64 Yju2 2.11 92.04 6.19 1.77 4.31 that can assesses the precise fitting of an atomic model into the map during refinement. To validate the model-to-map correctness of atomic models from cryo-EM, refinement should result in EMRinger scores above 1.0 for well-refined structures with maps in the 3- to 4-Å range. 10 Wan & Yan et al Table S4 Summary of model building, refinement and validation for RNA components of the spliceosomal C complex from S. cerevisiae. Model building software Coot & RCrane Refinement Software Phenix/Phenix.Erraser Validation Clash scores Correct sugar puckers (%) Good backbone (%) Good bonds (%) Good angles (%) All RNAs U6 snRNA U5 snRNA U2 snRNA Pre-mRNA 6.32 98.36 73.68 99.90 99.85 7.89 97.09 69.93 99.84 99.84 4.59 97.92 77.08 99.91 99.86 2.65 100.00 83.33 100.00 99.83 7.85 100.00 68.42 99.92 99.85 11 Wan & Yan et al Fig. S1 A flow chart for the cryo-EM data processing and structure determination of the spliceosomal C complex from S. cerevisiae. The final reconstruction has an average resolution of 3.41 Å. Please refer to Materials and Methods for details. This figure, together with Figs. S2A and S3, were prepared using CHIMERA (67). All other structural images were created using PyMol (68). 12 Wan & Yan et al Fig. S2 Cryo-EM analysis of a catalytic step I spliceosome (the C complex) from Saccharomyces cerevisiae (S. cerevisiae). (A) Representative 2D class averages of the spliceosomal C complex from S. cerevisiae. (B) The overall resolution is estimated to be 3.41 Å on the basis of the gold standard FSC criterion of 0.143. (C) An overall view of the EM density map for the C complex at an average resolution of 3.41 Å. The local resolutions are color-coded for different regions of the C complex. The surface view of the spliceosome is shown here. The resolution reaches 2.9-3.5 Å for the core regions of the C complex. (D) The EM density map is low-pass filtered to 10 Å resolution to show the more flexible regions of the C complex. 13 Wan & Yan et al Fig. S3 Cryo-EM analysis designed to improve the EM density maps of the peripheral regions of the spliceosomal C complex from S. cerevisiae. Starting from the 161,066 polished particles that yield an average resolution of 3.41 Å, we performed two more rounds of 3D classification. After the first round of 3D classification and auto-refinement, 80,367 particles of one major class yield a reconstruction with an average resolution of 3.65 Å. After the second round of 3D classification and auto-refinement, 20,686 particles give rise to a reconstruction with an average resolutions of 4.6 Å. In both cases, the resulting EM density maps show improved features for some regions of the C complex compared to the 3.41 Å reconstruction. 14 Wan & Yan et al Fig. S4 Cryo-EM analysis of the spliceosomal C complex from S. cerevisiae. (A) Angular distribution of the particles used for the reconstruction of the spliceosomal C complex at 3.65 Å resolution. Each cylinder represents one view and the height of the cylinder is proportional to the number of particles for that view. Two orientations of the C complex are shown. (B) FSC curves of the final refined model versus the overall 3.65 Å map it was refined against (black); of the model refined in the first of the two independent maps used for the gold-standard FSC versus that same map (red); and of the model refined in the first of the two independent maps versus the second independent map (green). The almost perfect overlap between the red and green curves indicates that the refinement of the atomic coordinates did not suffer from severe overfitting. (C) Estimation of the average resolution of the cryo-EM reconstructions on the basis of the gold standard FSC criteria of 0.143. Shown here are FSC curves for the overall reconstruction of all particles for the C complex (3.41 Å, blue line, 161,066 particles) and the reconstructions designed to visualize the more flexible regions of the C complex (3.65 Å, magenta line, 80,367 particles; 4.6 Å, cyan line, 20,686 particles). 15 Wan & Yan et al Fig. S5 EM density maps of Prp8 and Snu114 in the spliceosomal C complex. For the central spliceosomal component Prp8, the EM density maps are shown for the N-domain (A), RT Palm/Finger (B), Thumb/X (C), Linker (D), Endonuclease domain (E), RNaseH-like domain (F), and five representative secondary structural elements from these regions of Prp8 (G). For the only GTPase Snu114, the EM density maps are shown for the overall structure (H) and three representative α-helices (I). 16 Wan & Yan et al Fig. S6 EM density maps for the protein components of the C complex. Shown here are the density maps of the splicing factor Yju2 (A), two representative structural elements and a zinc finger of Yju2 (B), the splicing factor Cwc25 (C), the interface between Yju2 and Cwc25 (D), the NTR component Bud31 (E), a representative αhelix of Bud31 (F), the NTC component Cef1 (G), two representative secondary structural elements of Cef1 (H), the NTC component Syf2 (I), two α-helices of Syf2 (J), the NTC component Clf1 (K), and two representative α-helices of Clf1 (L). 17 Wan & Yan et al Fig. S7 EM density maps of protein components and their interfaces with surrounding proteins. (A) The EM density maps of the NTR component Prp46 and its interface with Snu114 (residues 67-97), Prp8 (residues 737-837), Cwc15 (residues 4-41), and Prp45 (residues 30-94). Three perpendicular views are shown. (B) EM density maps at the interface among Syf2, Cef1 (residues 163-252), the N-terminal half of Clf1, and U2 snRNA. (C) EM density maps at the interface among the NTR component Prp45 (residues 156-186), the Myb domain of Cef1, Syf2, and Prp8 (residues 737-837). (D) EM density maps at the interface among the Linker domain of Prp8, MA3 domain of Cwc22, Cwc21 (residues 2-28), and 5’-exon. Two perpendicular views are shown. 18 Wan & Yan et al Fig. S8 EM density maps of the RNA elements. (A) Overall EM density maps for the RNA elements at the center of the C complex. The four RNA molecules are colorcoded. The label “5’-intron” refers to the intron sequences at the 5’-end of the intron, including the 5’-splice site (5’SS) and the ensuing nucleotides. Two perpendicular views are shown. (B) Overall EM density maps for U5 snRNA. (C) Two close-up views on the EM density maps of loop I of U5 snRNA (upper panel) and a duplex region (lower panel). (D) A close-up view on the duplex between the 5’-exon sequences (red) and loop I of U5 snRNA (orange). 19 Wan & Yan et al Fig. S9 EM density maps of U6 snRNA and the catalytic center. (A) Overall EM density maps of U6 snRNA. (B) Two close-up views of the local EM density maps for the intramolecular stem loop (ISL) of U6 snRNA and helix II of the U2/U6 snRNA duplex. (C) Two perpendicular views of the EM density maps for the RNA elements at the catalytic center. The RNA elements include the ISL of U6 snRNA (green), loop I of U5 snRNA (orange), a small portion of U2 snRNA (marine), the 5’exon (red), and the intron lariat (magenta). 20 Wan & Yan et al Fig. S10 EM density maps of the active site. (A) A close-up view on the EM density maps at the active site. The two catalytic metals are shown in red spheres and the M1 magnesium ion (Mg2+) is coordinated by four ligands in a planar fashion. (B) A close-up view on the EM density maps centered around the invariant adenine nucleotide of the BPS. The 2’-OH group is covalently joined with the phosphate at the 5’-end of the 5’-splice site (5’SS). (C) A close-up view on the EM density maps centered around M1. The lariat junction and 5’-exon are shown. (D) A close-up view on the EM density maps for the lariat junction and the duplex between loop I of U5 snRNA and 5’-exon. 21 Wan & Yan et al Fig. S11 EM density maps of the protein components around the RNA elements at the catalytic center. (A) Two overall views on the EM density maps for Yju2, Cwc25, Isy1, Syf2, and a portion of Cef1. (B) A close-up view on the EM density maps of the splicing factor Yju2. (C) A close-up view on the EM density maps of the splicing factor Cwc25. (D) A close-up view on the EM density maps of the NTC component Isy1. (E) A close-up view on the EM density maps of a portion of the NTC component Cef1. (F) A close-up view on the EM density maps of the NTC component Syf2. 22 Wan & Yan et al Fig. S12 Structural features of the spliceosomal C complex. (A) The protein components at the periphery of the C complex. The three corners are marked by U5 Sm ring and 3’-end sequences of U5 snRNA, Msl1/Leal1/U2 Sm ring and 3’-end sequences of U2 snRNA, and the carboxyl-terminal sequences of Clf1. The RNA elements are shown to indicate the location of the catalytic center. (B) The protein components at the center of the C complex. At least 16 proteins have been identified around the catalytic center of the spliceosome. Two views are shown. Proteins shown in the left panel include Cef1, Clf1, Cwc2, Cwc15, Cwc25, Ecm2, Isy1, Prp45, Prp46, Syf2, and Yju2. Proteins shown in the right panel include Bud31, Cwc21, Cwc22, Prp8, and Snu114. 23 Wan & Yan et al Fig. S13 Structural comparison of individual RNA elements among the three spliceosomal complexes. (A) Structural comparison of U5 snRNA from the S. cerevisiae C complex, the S. cerevisiae Bact complex (42), and the S. pombe ILS complex (10, 11). (B) Structural comparison of U6 snRNA from the three spliceosomal complexes. (C) Structural comparison of U2 snRNA from the three spliceosomal complexes. (D) Structural comparison of the lariat junctions between the S. cerevisiae C complex and S. pombe ILS complex (10, 11). 24 Wan & Yan et al Fig. S14 The RNaseH-like domain stabilizes a mobile RNA element. (A) A closeup view on the RNaseH-like domain in the S. cerevisiae U4/U6.U5 tri-snRNP. Together with Prp3, Prp6, and Prp31, the RNaseH-like domain of Prp8 plays an important role in orienting the U4/U6 snRNA duplex. (B) A close-up view on the RNaseH-like domain in the S. cerevisiae Bact complex (42). It interacts with Hsh155 and Bud13 to stabilize placement of the intron sequences. (C) A close-up view on the RNaseH-like domain in the S. cerevisiae C complex. 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