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: [email protected]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
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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.
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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