www.sciencemag.org/cgi/content/full/science.aag0291/DC1

Supplementary Materials for

Structure of a yeast activated spliceosome at 3.5 Å resolution

Chuangye Yan, Ruixue Wan, Rui Bai, Gaoxingyu Huang, Yigong Shi*

*Corresponding author. E-mail: shi-lab@tsinghua.edu.cn

Published 21 July 2016 on Science First Release DOI: 10.1126/science.aag0291

This PDF file includes

Materials and Methods Figs. S1 to S23 Tables S1 to S4 Supporting References

Yan & Wan et al

Materials and Methods

Cef1-TAP tagging in S. cerevisiae

The reported cryo-EM structure of the S. pombe spliceosome likely reflects that of the

intron lariat spliceosomal (ILS) complex, because EM density for the 5’-exon is

largely absent (1). To isolate the spliceosomes at the early-to-intermediate stages, we

relied on an affinity purification protocol to screen five different protein tags,

including Brr2, Cef1, Hsh155, Prp8, and Snu114. For each protein tag, we

experimented with several different purification conditions mainly by altering the salt

concentration. In the end, Cef1 as the affinity tag gave rise to the best outcome in

terms of spliceosome yield and quality.

The carboxyl-terminus of Cef1 was TAP-tagged by PCR-based gene

targeting method using the plasmid pF6Aa-CTAP-HphMX6 as a PCR template. The

TAP tag comprises protein A and calmodulin binding peptide. The PCR product was

transformed into haploid Saccharomyces cerevisiae (S. cerevisiae) cells by the lithium

acetate method (2), allowing homologous recombination. Transformants were

selected on hygromycin B-YPD solid medium. Correct integration of the tag into the

genome was confirmed by PCR at the sequence level and by Western blots at the

protein level. The resulting strain carries a TAP tag and the HphMX6 marker at its

carboxyl-terminus.

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Purification of the spliceosomal complexes

Purification of the yeast spliceosomal complexes was carried out essentially as

described (3) (Fig. S1A). Briefly, 54 liters of S. cerevisiae were cultured in YPD

medium at 30 oC to an OD600 of 3.5~4. The cell pellets (~135 mL) were collected by

centrifugation and resuspended in Buffer A containing 20 mM HEPES-KOH, pH 7.9,

150 mM KCl, 1.5 mM MgCl2, and 20% glycerol. The cell suspension was dropped

into liquid nitrogen to form yeast beads with a diameter of 3-6 mm and pulverized to

powder by SPEX 6870 Freezer Mill. The frozen cell powder was thawed at room

temperature and resuspended in Buffer A in the presence of a protease inhibitor

cocktail containing final concentrations of 0.5 mM phenylmethylsulphonyl fluoride

(PMSF), 2 mM benzamidine, 2.6 μg/ml aprotinin, 1.4 μg/ml pepstatin and 5 μg/ml

leupeptin. The cell lysate was first centrifuged at 18,000g for 1 hour and the

supernatant was centrifuged again at 100,000g for 1 hour, yielding ~132 mL cell

extract and a pellet of cell debris. The supernatant was incubated with IgG

Sepharose-6 Fast Flow resin (GE Healthcare) and cleaved by TEV protease at 18 oC

for 1.5 hours in Buffer B containing 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1%

NP40, 1 mM DTT, and 0.5 mM EDTA. The eluent was supplemented with 2 mM

CaCl2 and loaded into calmodulin affinity resin (Stratagene). Finally, the complex

was eluted by the CEB buffer (10 mM Tris-HCl, pH 8.0, 75 mM NaCl, 1 mM

Mg(OAc)2, 1 mM imidazole, 0.01% NP40, 1 mM TCEP, 0.5 mM EGTA), and

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concentrated by Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-100 membrane

(Merck Millipore). The final eluent was analyzed by urea PAGE for RNA detection

and negative staining EM for particle intactness. The resulting complex was used for

sample preparation for cryo-EM imaging.

The purified sample contains a mixture of different spliceosomal complexes,

as judged by the presence of all five snRNA species (U2, U4, U5L, U5S, and U6) on

the urea PAGE gel (Fig. S1B) and the appearance of the particles by negative staining

EM (Fig. S1C). By applying the sample to 10-30% glycerol gradient centrifugation,

we were able to individually enrich the Bact and C complexes (Fig. S1D);

unfortunately, this additional purification step results in the loss and dissociation of

the majority of the spliceosomal complexes. A rough estimate indicates that only

about 30-40 percent of the original Bact or C complex remained intact after the

centrifugation step. Thus, although the additional purification step allows enrichment

of the individual spliceosomal complexes, the final yield is highly undesirable.

Because the different spliceosomal particles can be conveniently differentiated by

two-dimensional (2D) and three-dimensional (3D) classifications of the electron

microscopy (EM) data analysis, we decided not to apply this additional step of

purification. This strategy proved to be effective.

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EM data acquisition and processing

Uranyl acetate (2%, w/v) was used for negative staining. Briefly, the copper grids

supported by a thin layer of carbon film (Zhongjingkeyi Technology Co. Ltd) were

glow-discharged. 4 µl of the sample at a concentration of ~0.02 mg/ml were applied

onto the grid for 1 minute and stored at room temperature. Images were taken on an

FEI Tecnai Spirit Bio TWIN microscope operating at 120 kV to verify the sample

quality. The same carbon-coated copper grids as those used for negative staining were

used for cryo-EM specimen preparation. Cryo-EM grids were prepared with Vitrobot

Mark IV (FEI Company), using 8 oC and 100 percent humidity. Aliquots of 4 µl

sample at a concentration of ~0.27 mg/mL were applied to glow-discharged grids,

blotted for 2.5 seconds and plunged into liquid ethane cooled by liquid nitrogen.

Images were taken by an FEI Titan Krios electron microscope operating at 300 kV

with a nominal magnification of 22,500x. Images were recorded by a Gatan K2

Summit detector (Gatan Company) using the super-resolution mode, with a pixel size

of 0.653 Å. Defocus values varied from 1.6 to 2.6 μm. Each image was

dose-fractionated to 32 frames with a dose rate of ~8.2 counts/sec/physical-pixel

(~4.7 e-/sec/Å2), an total exposure time of 8.0 seconds, and 0.25 second per frame.

UCSFImage4 was used for all data collection (4).

Preliminary image processing

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A total of 12,142 cryo-EM micrographs were collected. All 32 frames in each image

were aligned and summed using the whole-image motion correction program

MOTIONCORR (5), with 2-fold binned to a pixel size of 1.306 Å. The anisotropic

magnification distortion of the micrographs collected on the FEI Titan Krios

microscope was estimated by the program “mag_distortion_estimate” and corrected

by the program “mag_distortion_correct” (6). The defocus value of each image was

determined by CTFFIND3 (7).

An initial data set of 841 micrographs were collected and processed to

inspect the sample quality and composition (Fig. S2). 124,646 particles were

semi-autopicked using the reference-based particle picking subroutine in RELION (8).

The templates for particle picking were obtained from the 2D class averages

calculated from ~3,000 manually picked particles. Particle sorting and two rounds of

reference-free 2D classification were performed to remove ice spots, contaminants,

damaged particles and aggregates using particles binned to a pixel size of 5.224 Å,

yielding 53,066 particles (Fig. S2).

Relying on the STAR file derived from 2D classification, all 53,066 particles

were read back to their original images based on their refined centers. This procedure

is the same as that employed in structure determination of a late-stage spliceosome

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from S. cerevisiae and a U4/U6.U5 tri-snRNP from S. pombe (1, 9). Examination of

the micrographs and comparison with the original semi-autopicked 124,646 particles

revealed three findings. First, most ice spots, contaminants, damaged particles and

aggregates have been successfully removed by prior classifications. Second, a sizable

fraction of the good particles appear to have been removed by prior classifications.

Third, some of the remaining particles are mis-centered or appear to be aggregates.

These considerations necessitated one round of manual particle picking and

discarding so as to improve the overall quality of the picked particles. One round of

manual particle picking and discarding was performed with these improved

coordinate files, yielding a final set of 55,772 particles for further processing (Fig. S2).

The published 3.6 Å spliceosome cryo-EM map was low-pass filtered to 40 Å by

CHIMERA (10) and used as the reference for 3D classification. Using binned

particles with a pixel size of 2.612 Å, one round of 3D classification was performed

by global search. As predicted from our biochemical characterization (Fig. S1), one

class of the particles, representing only about 8.3 percent of the total, display an

umbrella appearance that is similar to the reported EM structure of the Bact complex

(11). These particles are the subject of this study. In addition, another class,

representing about 34.5 percent of the total particles, exhibits an appearance that is

characteristic of the C complex or the ILS complex (Fig. S2). These particles are the

subject of an ongoing study that will be reported in the near future. Notably, the 3D

reconstruction of the Bact complex differs in a major way from that of the C complex

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or ILS complex by having a chuck of density on the periphery, which represent the

SF3b complex, the RES complex, and well-defined Brr2 (see later). The rest three

classes, representing 57.2 percent of the total, fail to display features that are

characteristic of the known spliceosomal complexes and are thus left out for

immediate investigation.

Image processing

The above-described procedure was applied to all 12,142 micrographs (Fig. S3). In

total, 1,304,231 particles were picked using the reference-based particle picking

subroutine in RELION (8). Particle sorting and two rounds of reference-free 2D

classification were performed to remove ice spots, contaminants, damaged particles

and aggregates, yielding 573,541 particles. All 573,541 particles were read back to

their original images based on their refined centers. One round of manual particle

picking and discarding was performed, resulting in the removal of 97,667 bad

particles and inclusion of 285,893 new particles. In the end, a data set of 761,767

particles was used for further processing.

Our preliminary analysis revealed that the spliceosomal Bact complex is only

represented by about 8.3 percent of the total particles (Fig. S2); thus finding all

particles that represent the genuine Bact complex is important to the improvement of

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data quality. Based on our experience, one round of 3D classification may be

insufficient for selection of all Bact particles into one class or Bact-related classes, and

some of the Bact particles might be classified into other spliceosomal complexes or

even other contaminating complexes such as the ribosome. Our 3D classification

results also show that different parameters often result in the classification of different

percentages for the Bact complex. To avoid the problem of discarding good particles

for the low-abundance complex, we simultaneously performed three independent 3D

classifications (K=5, 6, and 7) (Fig. S3). Then we merged all four classes that appear

to represent the Bact complex; using an in-house script, we removed the duplicated

particles according to the unique index of each particle given by RELION. This

procedure allowed us to generate 151,323 particles, representing 19.8 percent of the

total. This procedure was repeated one more time, resulting in 105,685 good particles

(13.9 percent of the total). The Bact complex was gradually enriched. A third round of

3D classification (K=4) yielded 84,486 particles in one major class, which have an

average resolution of 3.9 Å after auto-refinement. Following per-particle motion

correction and radiation-damage weighting (known as particle polishing) (12), these

polished particles give a reconstruction with an improved resolution of 3.58 Å after

auto-refinement. Finally, an additional round of 3D classification was performed

without alignment using the refined polished particles. 77,312 particles in one class,

or 91.5 percent of the input, yield a final reconstruction with an average resolution of

3.52 Å (Fig. S3). The angular distribution of the particles used for the final

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reconstruction of the Bact complex is reasonable, and the refinement of the atomic

coordinates did not suffer from severe overfitting (Fig. S4). The resulting density

maps show clear features for the secondary structural elements and amino acid side

chains for most protein components of the Bact complex (Figs. S5-S12). The RNA

elements and their interacting proteins are also well defined by the EM density maps

(Fig. S13-S16).

Reported resolutions are calculated on the basis of the gold-standard FSC

0.143 criterion, and the FSC curves were corrected for the effects of a soft mask on

the FSC curve using high-resolution noise substitution (13). 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 (14). Local resolution variations were estimated using ResMap

(15).

Model building and refinement

Due to a wide range of resolution limits for the various regions of the spliceosomal

Bact complex, we combined de novo model building and homologous structure

modeling to generate an atomic model (Table S1). Identification and docking of the

components of the Bact complex were facilitated by the published structures of the

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spliceosome at 3.6 Å resolution and the tri-snRNP at 3.8 Å resolution (1, 9). The

proteins and the corresponding PDB accession codes are summarized in Table S2:

3JCM for Prp8, Snu114, U5 RNA, U5 Sm ring and 5’-EXON of pre-mRNA; 3JB9 for

U6 snRNA, U2 snRNA, all proteins of NTC core complex, all proteins of

NTC-Related proteins, Prp17 and branch point sequence of pre-mRNA; 2KOA for

Rds3; 1CVJ for Hsh49; 2MKC for the RES complex; 2JKD for Pml1; 4C9B for

Cwc22; 2CSY for Cwc24;3BKP for Cwc27; and 2OZB for Prp2. The atomic

coordinates of Syf2 and part of Cef1 were obtained from the unpublished cryo-EM

structure of the spliceosomal C complex. These structures were docked into the

density map using COOT (16) and fitted into density using CHIMERA (10).

The atomic models of U5 snRNA, Prp8, Snu114 from the published

cryo-EM structure of the tri-snRNP (9) and the crystal structure of Brr2 (accession

code 5DCA (17)) were directly docked into the density maps and manually rebuilt

using COOT (16). The atomic model of Rds3, Pml1, and NTC-related (NTR) proteins

were generated by CHAINSAW (18) and the backbone was manually adjusted using

COOT (16). After that, automated model rebuilding was performed with RosettaCM

using the adjusted model as the template and the experimental cryo-EM density as a

guide (19-21). The Rosetta distance homology modeling function “RosettaCM” was

specifically designed to assist cryo-EM model building. In this step, we allowed

Rosetta to generate 10 models for each protein and selected the best model by

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individually comparing the models with the cryo-EM density maps. Then the

hydrogen atoms of the generated model were removed and model building was

further performed manually using COOT (16). An atomic model for Rse1 was first

predicted by I-TASSER server (22), and the model was further manually corrected

and rebuilt using COOT (16).

For those protein sequences that lack a homologue structure, de novo model

building was performed; this was summarized in Table S2 with a “De novo building”

annotation. These proteins include Hsh155, Cus1, Ysf3, Prp11, Bud13, Cwc21, and

parts of Cwc22 and Cwc24. 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 cryo-EM structures

of the ILS complex (1, 23) and the U4/U6.U5 tri-snRNP (9), reported secondary

structures, published base pairing specifics, and the relative sizes of the purine and

pyrimidine bases. The RNA sequences were manually built using COOT (16).

Pre-mRNA was de novo modeled on the basis of the EM density maps using COOT.

The RNA nucleotides, together with all protein components, were refined using

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REFMAC in reciprocal space (24). To further improve the geometries of the RNA

nucleotides, the RNA elements alone were adjusted using RCrane (25). The

conformations of the RNA components were further refined using phenix.erraser (26).

ERRASER is a Rosetta program for modeling RNA nucleotides into density.

On the basis of the EM density maps, we identified four metal ions that are

bound by nucleotides in the ISL of U6 snRNA. 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 (27-31). In contrast, K+ is

usually measured at 2.8–3.5 Å from the coordinating ligands (27, 28, 32). 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 individual protein was carried out using

phenix.real_space_refine application in PHENIX in real space (33) with secondary

structure and geometry restraints to prevent over-fitting. The final overall model was

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refined against the overall 3.52 Å map using REFMAC in reciprocal space (24), using

secondary structure restraints that were generated by ProSMART (34). 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 (35) (Fig. S4B).

Protein structures in the Bact complex were individually validated through

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 (36) . EMRinger scores were calculated as described (37). 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

well-refined structures with maps in the 3- to 4-Å range. The RNA nucleotides in the

Bact complex were validated directly by the Molprobity server, and the results are

shown in Table S4.

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Table S1 Cryo-EM data collection and refinement statistics.

Data collection

EM equipment FEI Titan Krios Voltage (kV) 300 Detector Gatan K2 Pixel size (Å) 1.306 Electron dose (e-/Å2) 45.6 Defocus range (µm) 1.6~2.6 Reconstruction Software RELION 1.4 Number of used Particles 77,312 Accuracy of rotation (˚) 0.462 Accuracy of translation (pixels) 0.31 Final Resolution (Å) 3.52 Model building software Coot, Rosetta, RCrane Refinement Software Phenix & Refmac Map sharpening B-factor (Å2) -84.6 Average Fourier shell correlation 0.865

R-factor 0.299

Model composition Protein residues 13,505 RNA nucleotides 357 GTP 1 ATP 1 Validation R.m.s deviations Bonds length (Å) 0.010 Bonds Angle (˚) 1.311 Ramachandran plot statistics (％)

Preferred 92.61 Allowed 5.49 Outlier 1.90

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Table S2 Summary of model building for the yeast Bact spliceosomal complex.

Molecule Length Domain/Region PDB code Modeling Resolution (Å)

U5 snRNP

U5 snRNA 214 28:183 3JCM Homology modeling 2.8~4.0

Prp8

2413

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)

3JCM

4BGD

Homology modeling

Homology modeling

2.8~4.0

~4.0

Snu114 984 67:975 3JCM Homology modeling 2.8~4.0

Brr2 2176 113:2163 5DCA Homology modeling 3.0~4.0

SmB1

SmD1

SmD2

SmD3

SmE1

SmF1

SmG1

196

146

110

101

94

86

77

Sm fold

Sm fold

Sm fold

Sm fold

Sm fold

Sm fold

Sm fold

3JCM

Rigid docking

4.0~6.0

U6 snRNP U6 snRNA 112 nt 1:106 3JB9 Homology modeling 2.8~4.0

U2 snRNP

U2 RNA 1175 nt 1:66 3JB9 Homology modeling 2.8~4.0

Rse1 1361 WD40 domain (56:1361) - I-TASSER/Modeling 3.0~3.8

Hsh155 971 N-terminal Domain (16:74)

HEAT Repeat domain (156:971)

-

-

De novo building

De novo building

3.0~3.5

3.0~3.5

Cus1 436 131:289 - De novo building 3.0~4.0

Rds3 107 PHF domain (2:104) 2K0A Homology modeling 3.0~3.5

Ysf3 85 1:84 - De novo building 3.0~3.5

Hsh49 213 2 RRM domains Rigid docking ~8

Prp9 530 - - Not modelled -

Prp11 266 Zinc Finger domain (1:108) - De novo building 3.0~3.5

Prp21 280 - Not modelled -

RES complex

Bud13 266 213:265 2MKC De novo building 3.0~4.2

Pml1 204 N-terminal Domain (20-42)

Forkhead associated domain

2MKC

2JKD

Homology modeling

Rigid docking

3.5~4.2

4.0~5.0

Ist/Snu17 194 RRM_ist3 like domain 2MKC Homology modeling 3.5~4.2

NTC/Prp19

Complex

Clf1 687 TPR domain (40:275)

-

3JB9

Homology modeling

Rigid docking

3.0~4.5

~30

Syf1 859 - Rigid docking ~30

Cef1 590 Myb Domain (9:111)

-

Homology modeling

Rigid docking

2.8~3.5

~30

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Prp19 503 - Rigid docking ~30

Syf2 215 92:215 From C complex Rigid docking ~7

Snt309 175 - 3JB9 Rigid docking ~30

NTC-Related

proteins

Bud31 157 1:157 3JB9 Homology modeling 3.0~3.5

Cwc2 339 1:261 3JB9 Homology modeling 3.0~4.0

Cwc15 175 3:41/127:175 3JB9 Homology modeling 3.0~4.0

Ecm2 364 RRM domain (3:288) 3JB9 Homology modeling 3.0~5.0

Prp45 379 34:350 3JB9 Homology modeling 3.0~4.0

Prp46 451 WD40 domain (111:447) 3JB9 Homology modeling 2.8~3.5

Known

Splicing

Factors

Cwc21 135 2:28 - De novo building 3.0~3.5

Cwc22 577 MIF4G domain (11:263)

MA3 domain (279:485)

4C9B

-

Rigid docking

De novo building

5~10

3.0~3.5

Cwc24 259 Zf-CCCH Domain (126:169)

RING domain

-

2CSY

De novo building

Homology modeling

2.8~3.2

3.5~4.5

Cwc27 301 Cyclophilin domain (4:170) 3BKP Homology modeling 3.0~4.0

Prp2 876 2OZB Rigid docking 5~15

Spp2 185 - - Not modelled -

Yju2 278 - - Not modelled -

Step2

proteins

Prp17 455 50:75 3JB9 Homology modeling 3.0~4.2

Pre-mRNA Pre-mRNA - 61 nt - De novo building 2.8~4.2

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Table S3 Summary of model validation for individual proteins of the yeast Bact 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 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.

Molecule Molprobity Scores

Ramachandran plot statistics (％) EMRinger* Score Preferred Allowed Outlier

Prp8 2.06 93.00 5.72 1.28 3.67 Snu114 2.13 92.09 6.54 1.38 3.39

Brr2 2.09 94.24 5.04 0.71 1.89 Rse1 2.33 91.15 6.96 1.89 2.88

Hsh155 1.99 94.83 2.87 2.30 3.57 Cus1 2.16 94.56 5.44 0.00 2.47 Rds3 2.83 86.14 11.88 1.98 4.43 Ysf3 2.00 90.24 8.54 1.22 4.75 Prp11 1.94 92.63 6.32 1.05 2.98 Bud13 2.09 94.12 5.88 0.00 2.71 Pml1 2.02 95.78 3.61 0.60 0.85

Ist/Snu17 2.51 94.07 3.70 2.22 3.24 Clf1 2.09 94.87 5.13 0.00 1.72 Cef1 2.00 93.91 4.35 1.74 4.09

Bud31 2.01 91.61 6.45 1.94 3.00 Cwc2 2.22 93.44 4.25 2.32 2.43

Cwc15 1.79 89.23 10.77 0.00 4.78 Ecm2 2.27 88.14 10.73 1.13 1.57 Prp45 2.02 90.34 7.14 2.52 2.93 Prp46 2.20 90.45 7.46 2.09 3.28 Cwc22 1.85 92.68 5.85 1.46 3.45 Cwc24 2.53 87.16 11.01 1.83 2.86 Cwc27 2.27 89.04 8.90 2.05 3.13

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Table S4 Summary of model building, refinement and validation for RNA components of the yeast Bact spliceosome complex.

Model building Software Coot & RCrane Refinement Software Phenix/Phenix.Erraser

Validation Molprobity Server

Validation All RNAs U6 snRNA U5 snRNA U2 snRNA Pre-mRNA

Clash scores 4.86 1.82 2.15 3.85 8.55 Correct sugar puckers (%) 99.44 100.00 99.15 100.00 98.59 Good backbone conf. (%)

80.11 85.44 82.05 86.36 63.38

Good bonds (%) 99.98 100.00 100.00 99.94 100.00 Good angles (%) 100.00 100.00 100.00 100.00 100.00 The percentages of correct sugar puckers, good backbone conformations, good angles, and good bonds were calculated by subtracting the percentages of Probably Wrong sugar puckers, Bad backbone conformations, Bad angles, and Bad bonds reported in the MolProbity server, respectively, from 100 percent.

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Fig. S1 Purification and characterization of the spliceosomal complexes from Saccharomyces cerevisiae (S. cerevisiae). (A) A cartoon diagram of the purification protocol. The protein Cef1 was tagged by protein A and a calmodulin binding peptide. (B) The affinity-purified sample contained a mixture of different spliceosomal complexes. Shown here is a denaturing Urea-PAGE gel stained by SYBR® Gold. At least five major RNA species are clearly present, with their sizes corresponding to those of U6 snRNA (112 nucleotides), U4 snRNA (160 nucleotides), two forms of U5 snRNA (179 and 214 nucleotides), and U2 snRNA (1175 nucleotides). (C) A representative electron microscopy (EM) micrograph of the affinity-purified sample stained by uranyl acetate. At least three different spliceosomal complexes are present. Scale bar, 100 nm. (D) Two representative negative-stained EM micrographs of the sample that had been purified through one round of 10-30% glycerol gradient centrifugation. Scale bar, 100 nm. The spliceosomal Bact complex (right) and the C complex (left) had been greatly enriched, but the concentration and final yield for these complex became unacceptably low. We decided to directly use the affinity-purified sample for cryo-EM data acquisition and to rely on two-dimensional (2D) and three-dimensional (3D) classifications to separate the different spliceosomal complexes.

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Fig. S2 Analysis of the initial data set of 841 micrographs of the

affinity-purified spliceosomal complexes from S. cerevisiae. This analysis shows

that a small proportion of the particles (about 8.3 percent) corresponds to the Bact

complex. Thus enriching the Bact complex is important for data processing. Please

refer to Materials and Methods for details. This figure, together with Figs. S3 and S4A,

were prepared using CHIMERA (10). All other structural images were created using

PyMol (38).

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Fig. S3 A flow chart for the cryo-EM data processing and structure determination of the spliceosomal Bact complex from S. cerevisiae. The final reconstruction has an average resolution of 3.52 Å. Please refer to Materials and Methods for details.

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Fig. S4 Cryo-EM analysis of the spliceosomal Bact complex from S. cerevisiae.

(A) Angular distribution of the particles used for the final reconstruction of the

spliceosomal Bact complex. 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 Bact complex are shown. (B) FSC curves of the final refined model versus the

overall 3.52 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 little difference between the red and green curves

indicates that the refinement of the atomic coordinates did not suffer from severe

overfitting.

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Fig. S5 EM density maps for Prp8 in the spliceosomal Bact complex. Shown here are EM density maps for the N-domain (A), RT Palm/Finger (B), Thumb/X (C), Linker (D), endonuclease domain (E), RNaseH-like domain (F), and representative secondary structural elements from these regions of Prp8 (G). The side chain features for many residues are clearly visible, allowing assignment of specific amino acids.

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Fig. S6 EM density maps for Snu114 (Cwf10 in S. pombe) and Brr2. (A) Overall EM density maps for Snu114. (B) EM density maps for 12 representative secondary structural elements. Bulky residues are labeled. (C) Overall EM density maps for Brr2. (D) EM density maps for four α-helices and two β-strands of Brr2.

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Fig. S7 EM density maps for Rse1 and Rds3. (A) Overall EM density maps for Rse1. It contains three β-propellers, each comprising seven WD40 repeats. (B) EM density maps for 14 representative secondary structural elements of Rse1. The quality of the density maps allowed de novo modeling of Rse1. (C) Overall EM density maps for Rds3. (D) EM density maps for two representative regions of Rds3.

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Fig. S8 EM density maps for Hsh155, Ysf3, and Cus1. (A) Overall EM density maps for Hsh155. (B) EM density maps for 18 representative α-helices of Hsh155. The quality of the density maps allowed de novo modeling of Hsh155. (C) Overall EM density maps for Ysf3. (D) EM density maps for two representative α-helices of Ysf3. (E) Overall EM density maps for Cus1. (F) EM density maps for four representative secondary structural elements of Cus1. The density maps allowed de novo modeling of Cus1.

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Fig. S9 EM density maps for the retention and splicing (RES) complex. (A) Overall EM density maps for the RES complex. The RES complex comprises Pml1, Bud13, and Snu17. The quality of the density maps allowed de novo modeling of these three proteins. The NTR component Prp45 interacts with both Pml1 and Bud13 to stabilize the RES complex. EM density maps of the four boxed regions are shown in panels B through E. (F) EM density maps for Bud13 and a representative α-helix. (G) EM density maps for Snu17 and a representative α-helix.

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Fig. S10 EM density maps for Prp45, Prp46, and Clf1. (A) Overall EM density maps for the highly extended protein Prp45. (B) EM density maps for seven representative secondary structural elements of Prp45. (C) Overall EM density maps for the β-propeller protein Prp46. (D) EM density maps for nine representative β-strands of Prp46. (E) Overall EM density maps for the HAT repeat protein Clf1 (Syf3 in S. pombe). (F) EM density maps for six representative α-helices of Clf1.

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Fig. S11 EM density maps for Bud31, Cwc2, Ecm2, and Prp11. (A) Overall EM density maps for Bud31. (B) EM density maps for four local structural elements of Bud31. (C) Overall EM density maps for Cwc2. (D) EM density maps for six local structural elements of Cwc2. (E) Overall EM density maps for Ecm2. (F) EM density maps for two representative local structural elements of Ecm2. (G) Overall EM density maps for the SF3a component Prp11 (SF3a66 in human). (F) EM density maps for two representative α-helices of Prp11.

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Fig. S12 EM density maps for the splicing factors Cwc22, Cwc24 and Cwc27, and the NTC component Cef15 (Cdc5 in S. pombe). (A) Overall EM density maps for Cwc22. (B) EM density maps for four representative α-helices of Cwc22. (C) Overall EM density maps for Cwc24. (D) EM density maps for four local structural elements of Cwc24. (E) Overall EM density maps for Cwc27. (F) EM density maps for two representative local structural elements of Cwc27. (G) Overall EM density maps for Cef1. (H) EM density maps for five representative α-helices and one loop of Cef1.

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Fig. S13 EM density maps of the RNA elements. (A) Overall EM density maps for the RNA elements. The four RNA molecules are color-coded. 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 (left panel) and its base-pairing interactions with the 5’-exon sequences (right panels). (D) Two close-up views on the EM density maps of the duplex regions of U5 snRNA. (E) Three close-up views on the EM density maps of U5 snRNA.

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Fig. S14 EM density maps of U6 snRNA and the active site. (A) Overall EM density maps for U6 snRNA. (B) Two close-up views of the local EM density maps of U6 snRNA. (C) Two perpendicular views of the EM density maps of the intramolecular stem loop (ISL) of U6 snRNA and Helix I of the U2/U6 duplex. (D) A close-up view on Helix I of the U2/U6 duplex. (E) A close-up view on the RNA triplex between U2 and U6 snRNA. (F) A close-up view on the structural Mg2+ ions that help stabilize the ISL of U6 snRNA and its surrounding nucleotides. (G) A close-up view on the duplex between ACAGA box and the 5’SS of the intron. (H) A close-up view on the loop I base-pairing interactions with 5’-exon sequences. (I) Two close-up views of the active-site magnesium ions and their surrounding structural elements.

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Fig. S15 EM density maps of U2 snRNA and the surrounding structural components. (A) EM density maps of the duplex between U2 snRNA and the branch point sequence (BPS) of the intron. (B) Two close-up views of the local EM density maps of U2 snRNA. (C) An overall view on how the intron is bound by U2 snRNA and the protein components Hsh155 (yellow) and Rds3 (brown). (D) A close-up view on the EM density maps surrounding the invariant adenine nucleotide in the BPS. A number of residues from both Hsh155 and Rds3 together form a pocket to recognize the adenine nucleotide. (E) Two views of the EM density maps on the interactions among the RES complex, the SF3b complex, and the NTR protein Prp45. The RES complex, positioned at the bottom of Hsh155, directly recognizes the exposed intron sequences just outside of the SF3b complex. Prp45 closely interacts with the RES complex.

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Fig. S16 EM density maps at the active site. (A) An overall view of the EM density maps at the active site region, four surrounding proteins are shown here: Cwc24 (yellow), Cus1 (cyan), Prp11 (forest), Prp8 (magenta). (B) A close-up view of the EM density maps surrounding the invariant guanine nucleotide at the 5’-end of the 5’SS. The guanine base is surrounded by residues from the amino-terminal zinc finger domain of Cwc24, particularly Tyr155, Lys160, and Phe161. The zinc ion is located close to the guanine base. (C) A close-up view of the catalytic magnesium ion. An amino-terminal loop from Prp11 is positioned close to the active site. Two positively charge residues Lys10 and Lys11 are shown here. (D) A close-up view on the EM density maps of the active site and base-pairing of 5’-exon and loop I of U5 snRNA. Note the presence of a Tyr residue from Prp11. (E) A close-up view on the local EM density maps of the protein components that shape the active site. The four protein components are Prp11, Cwc24, Cus1, and the 1585 loop of Prp8, which closely interact with each other. (F) A close-up view on the local EM density maps of Cus1 around the active site. Two positively charged residues Arg230 and Lys226 are H-bonded to the nucleotides A51 and A52 of U6 snRNA.

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Fig. S17 Overall structure of the spliceosomal Bact complex. In the structure,

five subcomplexes are color-coded: orange for U5 snRNP, marine for U2 snRNP,

grey for NTC, cyan for NTR, and red for the RES complex. The splicing factors Prp2,

Cwc21, Cwc22, Cwc24, and Cwc27 are colored purple. Four views are shown.

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Fig. S18 Structural comparison of Prp8 and Spp42. The most highly conserved and the largest spliceosomal component is Prp8 in S. cerevisiae or Spp42 in S. pombe. Shown here are Prp8 from the U4/U6.U5 tri-snRNP (left panels), Prp8 from the Bact complex (middle panels), and Spp42 from the ILS complex (right panel). Compared to Prp8 in the tri-snRNP, the N-domain of the Bact complex and the N-domain of Spp42 are similarly moved closer to the core (represented by double-headed arrows).

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Fig. S19 Structures of the protein components in the Bact complex. (A) Structure of the SF3b central scaffold component Hsh155 in two perpendicular views. (B) Structure of Hsh155 with Ysf3, Cus1, and Prp11 bound. (C) Structure of Rse1 in two views. (D) Interactions within the SF3b complex. In this representation, the starting point is the structure of Rse1 bound to Ysf3, Cus1, Prp11, and Rds3. The three components of the RES complex are Snu17 (E), Pml1 (F), and Bud 13 (G). (H) Structure of the splicing factor Cwf27. (I) Structure of the ATPase/helicase Brr2. (J) Structure of the ATPase/helicase Prp2. (K) Structure of the splicing factor Cwf22. (L) Structure of the splicing factor Cwf24.

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Fig. S20 The splicing factors Cwc21. Cwc22, Cwc24, and Cwc27 and the

ATPase/helicase Prp2. (A) Cwc21 and the MA3 domain of Cwc22 bind to Prp8

and are both located close to the 5’-exon. Cwc21 forms a β-sheet with the Switch

loop of Prp8 and directly interacts with 5’-exon. (B) The RING domain of Cwc24,

containing two zinc fingers, is bound to Rse1 of the SF3b complex. Another zinc

finger domain at the amino-terminus of Cwc24 is located at the active site and

coordinates the bases GU in the 5’SS. (C) Cwc27, which is a peptidyl-prolyl

cis-trans isomerase (39), binds both the HLH domain of Brr2 and the endonuclease

domain of Prp8. (D) The ATPase/helicase Prp2, which mediates the structural

rearrangement of the Bact to B* complex, is bound to Hsh155 and located at the close

proximity of the RES complex and the 3’-end sequences of the intron.

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Fig. S21 Structural comparison of the RNA elements between the U4/U6.U5 tri-snRNP and the Bact complex. (A) Overall views of the RNA map in the Bact complex (left panel) and in the U4/U6.U5 tri-snRNP (9) (right panel). (B) Alignment of the RNA elements between the U4/U6.U5 tri-snRNP (9) and the Bact complex. The alignment was performed on the two U5 snRNA molecules (left panel). A close-up view focusing on the pre-mRNA molecules is shown in the right panel. In the tri-snRNP structure (9), the 5’-exon sequences of the pre-mRNA are already bound to loop I of U5 snRNA, and the 5’SS of the pre-mRNA is recognized by the ACAGA box of U6 snRNA. In the Bact complex, the 5’-exon sequences of the pre-mRNA are similarly bound to loop I of U5 snRNA, and the 5’SS of the pre-mRNA is similarly recognized by the ACAGA box of U6 snRNA.

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Fig. S22 Comparison of the catalytic centers between the spliceosomal Bact complex from S. cerevisiae and the pre-catalytic self-splicing group IIC intron. (A) Structure of the catalytic center of the spliceosomal Bact complex from S. cerevisiae, with a close-up view on the active site. The 5’-exon is paired with loop I of U5 snRNA. Mg2+ shown here is likely a catalytic metal (M2) and is coordinated by A59 and G60, which are part of the RNA triplex. The 5’SS forms a kink in the backbone that presents the scissile phosphodiester bond of the splice site to the active site. (B) Structure of the catalytic center of the self-splicing group IIC intron from Oceanobacillus iheyensis in the pre-catalytic state (40), with a close-up view on the active site. The sequences corresponding to 5’-exon is paired with EBS1. The only Mg2+ ion near the active site is not a catalytic metal ion. Similar to the Bact complex, the backbone is kinked at the junction of 5’-exon and the splice site. This RNA conformation represents that just prior to catalysis. Notably, predictions for the active site of the spliceosome was first proposed on the basis of structural studies of the group II introns (41, 42). Comparison between the spliceosomal Bact complex and the self-splicing group IIC intron was performed in PyMol (38).

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Fig. S23 Zinc-binding sites in the Bact complex are shown for Rds3 (A), Prp11

(B), Bud31 (C), Cwc2 (D), Ecm2 (E), and Cwc24 (F). In our structure, Rds3

contains three C4-type zinc fingers, Prp11 has a C2H2-type zinc finger, and Bud31

contains three zinc ions coordinated by nine Cys residues. Cwc2 contains a

C3H1-type zinc finger, Ecm2 has two C4-type zinc fingers, and Cwc24 contains three

zinc fingers: two C3H1 and one C4.

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