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1 Structure of a tyrosyl-trna synthetase splicing factor bound to a group I intron RNA Paul J. Paukstelis 1, Jui-Hui Chen 2, Elaine Chase 2, Alan M. Lambowitz 1,*, and Barbara L. Golden 2,*,. 1 Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, and Section of Molecular Genetics and Microbiology, School of Biological Sciences, University of Texas at Austin, Austin, TX 78712; 2 Department of Biochemistry, Purdue University, West Lafayette, IN *Contributed equally Correspondence: lambowitz@mail.utexas.edu and barbgolden@purdue.edu 1

2 Supplementary Notes Care was taken to minimize potential model bias from molecular replacement and to validate the accuracy of the refined structure. First, simulated annealing composite omit maps calculated in CNS 1 showed an outstanding fit to the refined structure (Supplementary Fig. 1). The simulated annealing composite omit map density masked by the four complexes was averaged using the NCS matrices. The overall density correlation between these four masked regions was Second, following the rigid-body refinement there was strong negative F o -F c density for the improperly fit P6a helix in the search model, as well as positive density corresponding to the proper fit (Supplementary Fig. 7). Thus, any model bias from molecular replacement was not pervasive enough to prevent recognition of an incorrectly positioned region. Third, we generated large-scale omit maps by deleting entire protein subunits or RNA domains from one complex in the asymmetric unit and then recapitulating the omitted regions in the subsequent F o -F c density (Supplementary Fig. 8). Fourth, there was strong difference-density for regions in both the RNA and protein that were not present in the molecular replacement search models. Difference-density was present for regions of the protein that were disordered in the initial CYT-18/ structure 2 (Supplementary Fig. 9a-c; seven N-terminal amino acids, an eleven amino acid loop (Arg147 to Ser157), and seven C-terminal amino acids). We attempted to model these regions, but due to a lack of restraints and the fact that all are loop regions, they showed poor backbone geometry when refined and were omitted from the final model. We were, however, able to build an additional seven nucleotides of the J5/5a and P5a regions that were disordered in the initial Twort crystal structure 3. Electron density corresponding to the intermolecular contacts involving the L5 regions (A67-A74) of two RNAs within the asymmetric unit was strong, but insufficient to provide an unambiguous model of L5 (Supplementary Fig. 9d). To verify that model bias would not prevent recognition of key features, we repeated the entire structure determination, starting with molecular replacement, using search models with the H0 α- 2

3 helix deleted from the protein and the P7 helix deleted from the RNA. Supplementary Fig. 10 shows F o -F c density for these deleted regions at different stages of the refinement. Model bias cannot be responsible for this difference-density, as these portions of the model were not used for phase calculations at any point during the structure determination. Finally, the structure is consistent with distances derived from sitedirected cleavage from eleven amino acid positions. These data were used previously to derive a model for the complex by rigid body docking 2 whose overall geometry agrees very well with that derived here from the diffraction data (Supplementary Fig. 3). We note that the R-factors for this structure are lower than most structures determined at this resolution. This is likely due to two reasons. First, the models that were used for molecular replacement and refinement were not homology models, but refined structures of exactly the same molecules present in complex. These high-resolution structures provide a much more accurate model of the complex than a structure built de novo from low resolution data or from a homology model. Second, the multiple complexes in the asymmetric unit were highly restrained using four-fold non-crystallographic symmetry averaging and we applied distance restraints derived from the component structures. Together these restraints significantly improve the parameter-to-observation ratio that is problematic in low-resolution refinement. 3

4 Supplementary Table S1 Data collection and refinement statistics Twort/CYT-18 Data collection Space group P2 Cell dimensions a, b, c (Å) 231, 123, 235 α, β, γ ( ) 90.0, 90.3, 90.0 Resolution (Å) ( ) * R merge 7.4 (32.3) Range (Å) I/σI Redundancy Completeness (%) Overall ( ) Refinement Resolution (Å) ( ) No. reflections 66,048 a R work/ R free 23.5 (36.4)/ 24.9 (41.2) No. atoms 44,336 Protein RNA Atomic parameters 177,344 Atomic restraints NCS 133,008 Defined b 3476 B-factors Protein RNA R.m.s deviations Bond lengths (Å) Bond angles (º) 1.22 *Highest resolution shell is shown in parenthesis. a 5% of the total reflections were excluded for cross-validation b Defined restraints include protein secondary-structure and RNA hydrogen bonding and base-pairing restraints. The structure was determined from data derived from a single crystal. 4

5 Supplementary Table 2. Molecular replacement statistics from Phaser * Search model LLG Z-score (rot) Z-score (trans) Packing Protein dimer Protein dimer Protein dimer Protein dimer RNA RNA RNA RNA * R.m.s deviation set to 1.5 Å for both components. 5

6 Supplementary Table 3. Summary of interactions between CYT- 18/ and Twort intron RNA Secondary structural element and contacted nucleotides a J3/4 P4[5'] P5/P5a[5'] P4[3'] P6[5'] A47, U48 U49-G53 A63-C65 A89, G90 G90, C91 Contacting amino acids b Lys40 *, Tyr41, Lys44 *, Trp190 Tyr41, His201, Lys205 *, Gln261, Gln262 His167-A, Ile196-A, His201-A His167, Lys171 *, Gly195, Ile196 Lys44 *, Lys171 *, Lys193 *, Arg194 *, Gly195 Supporting references 2, 4, 5 2, 6, 7, 8, 9, 10 2, 6, 7, 9, 11, 10, 12 2, 6, 7, 8, 9, 11, 12 6, 7, 8, 9, 11, 10 P6a[3'] A108-G111 Lys172 *, Glu175, Thr179, Arg182 * 2, 6, 7, 9 P7[5'] A123, C124 Pro39, Lys40 * 2, 5, 6, 7, 9, 11 P9[5']/L9 A202, U203, G205, A206 Pro292, Asp293, 2, 6, 7, 9, 11, Pro294, Gln295 10, 12 P9[3'] A212 Gln295 2, 6, 7, 9, 11, 10 a Contacts are defined as RNA and protein atoms within 4 Å. b Amino acid residues are from subunit B, unless followed by "-A" to denote subunit A. Amino acid residues in CYT-18-specific insertions H0, Ins1, and Ins2, are in cyan. *Previously identified as basic amino acid substitution relative to non-splicing bacterial TyrRSs

7 Supplementary Figure 1 Electron density. a, NCS-averaged sigmaa-weighted 2m F o - D F c simulated annealing composite omit map electron density for one of four complexes in the asymmetric unit contoured at 1.0 σ. The density masked by the four complexes in the asymmetric unit was averaged after map calculation and had an overall correlation of b and c, steroviews showing a close up of the electron density around the P4-P6 junction and around the Ins2-B interaction with P9, respectively. 7

8 Supplementary Figure 2 The conformations of CYT-18 protein and the Twort ribozyme change little upon binding. The structures of a, CYT-18/ and b, Twort ribozyme in the cocrystal (green) are compared with the individual crystal structures (orange; PDB accession numbers 1Y42 and 1Y0Q) 2,3. The r.m.s. deviations of all observable Cα atoms in CYT-18 subunits A and B are and Å, respectively. For the intron, P6a and P9.1, which are sites of crystal contacts, show significant displacement from their positions in the crystal structures of the unbound RNA. Excluding these regions, the bound Twort RNA has an r.m.s. deviation of 1.56 Å for 192 nucleotides (all atoms) relative to the unbound RNA. 8

9 Supplementary Figure 3 Comparison of the cocrystal structure with a previous model derived using biochemical data. The cocrystal structure described here for the complex between CYT-18/ and the Twort group I intron ribozyme (panel a) closely resembles the model for CYT-18/ bound to the N. crassa ND1 intron derived by rigid-body docking using distance restraints from 92 site-directed hydroxyl radical cleavages of the ND1 RNA generated by EPD-Fe(II) conjugated at eleven positions in the protein monomer 2 (panel b). The P4-P6 domains of the RNAs are shown in blue and the P3-P9 domains are shown in green. 9

10 Supplementary Figure 4 CYT-18 binds group I introns on a surface distinct from that which binds trna Tyr. trna Tyr (orange) was docked onto the active site of CYT-18 (magenta and violet) based on the Thermus thermophilus TyrRS/tRNA Tyr cocrystal structure (PDB accession number 1H3E) 13. The acceptor arm (AA), D-loop (D), anticodon loop (AC) and variable loop (V) are labeled. The intron RNA binds on the side opposite trna Tyr with each of the CYT-18-specific insertions H0, Ins1, and Ins2 (cyan) contributing to intron RNA-binding. 10

11 Supplementary Figure 5 Stereoviews showing CYT-18 binding to the P4-P6 domain. a, The P4-P6 junction binds in a cleft formed by β-strand D of subunit B (βd-b; Lys193, Arg194) and α-helix H5 of subunit B (H5-B; His167, Lys171). The protein may also contact the 5'-strand of P4 using helices H6-B and H10-B (His201, Gln261). b, Side chains from H5-B and Ins1-B bind in the major groove of P6 and P6a and may help establish the correct conformation of J6/6a for P3 docking in the minor groove. c, P5a A64 and C65 bind in a pocket formed by βd and H5 in subunit A. The same pocket in subunit B is used to bind the P4-P6 junction (see panel a). 11

12 Supplementary Figure 6 Stereoviews showing interactions of CYT-18-specific insertions. a, H0-B and Ins1-B Trp190 create a binding pocket for J3/4, with potential for ionic contacts between Lys40 and Lys44 and A47 and U48. Tyr41 is positioned between U48 and U49 and may induce an upward kink in the phosphate of U49 (blue sphere), forcing the U48 ribose into the P6 minor groove for tertiary contacts with U116. H0-B likely contacts P7, the guanosine-binding site, at A123 and C124. b, Ins2-B provides a platform for docking P9, helping position the L9-tetraloop for docking in the minor groove of P

13 Supplementary Figure 7 Stereoview of positive and negative F o -F c density for P6a following molecular replacement. The molecular replacement solution is shown as the orange ribbon, the blue ribbon is the final refined model. Positive (green) and negative (magenta) density is shown contoured at the 2σ level. The positive density extending downward from P6a corresponds to P9.1 from a symmetry-related molecule. Both P6a and P9.1 are shifted from the molecular replacement solution to accommodate this crystal contact. 13

14 . Supplementary Figure 8 Stereoviews of large-scale omit map F o -F c electron density contoured at 3 σ. Following refinement, regions in red were omitted from the model and used to calculate F o -F c density. These regions correspond to a, CYT-18 subunit B, b, CYT-18 subunit A, and c, Twort P4-P6 domain (nucleotides ). 14

15 Supplementary Figure 9 Stereoviews of F o -F c density for portions of the molecules not present in the molecular replacement search models. Difference density is clearly present for several regions of the molecules not modeled in the initial crystal structures due to disorder. Both F o -F c density (green) contoured at 3σ and 2F o -F c density (blue) contoured at 1.5σ are shown. The panels correspond to a, residues from protein subunit B, b, 7 C-terminal amino acids of subunit B, c, 7 amino acids from the N- terminus, and d, the interaction of two L5 loops from two RNA molecules in the asymmetric unit. 15

16 Supplementary Figure 10 Stereoviews of F o -F c density (3σ) for regions corresponding to H0 and P7 during omit refinement. Regions in red were deleted from the molecular replacement search models and were not present at any point during the refinement and are shown here for reference. Blue ribbons show the model that was refined with these regions omitted (P9.1 and P7.2 are not shown for clarity). The panels represent a, after rigid-body refinement following molecular replacement, b, after the first refinement, but before building P5a or correction of P6a and P9.1, and c, after refinement following the building of P5a and correction of P6a and P9.1. Density in the oval corresponds to unmodeled N-terminal residues (see Supplementary Fig. 9c). 16

17 Supplementary Figure 11 Ramachandran plot. Ramachandran plot for all protein residues in the asymmetric unit as determined with Procheck

18 Supplementary Information References 1. Brünger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, (1998). 2. Paukstelis, P. J. et al. A tyrosyl-trna synthetase adapted to function in group I intron splicing by acquiring a new RNA binding surface. Mol. Cell. 17, (2005). 3. Golden, B. L., Kim, H. & Chase, E. Crystal structure of a phage Twort group I ribozyme-product complex. Nat. Struct. Mol. Biol. 12, 82-9 (2005). 4. Waldsich, C., Grossberger, R. & Schroeder, R. RNA chaperone StpA loosens interactions of the tertiary structure in the td group I intron in vivo. Genes Dev. 16, (2002). 5. Myers, C. A. et al. A tyrosyl-trna synthetase suppresses structural defects in the two major helical domains of the group I intron catalytic core. J. Mol. Biol. 262, (1996). 6. Caprara, M. G., Lehnert, V., Lambowitz, A. M. & Westhof, E. A tyrosyl-trna synthetase recognizes a conserved trna-like structural motif in the group I intron catalytic core. Cell 87, (1996). 7. Caprara, M. G., Mohr, G. & Lambowitz, A. M. A tyrosyl-trna synthetase protein induces tertiary folding of the group I intron catalytic core. J. Mol. Biol. 257, (1996). 8. Chen, X., Gutell, R. R. & Lambowitz, A. M. Function of tyrosyl-trna synthetase in splicing group I introns: an induced-fit model for binding to the P4-P6 domain based on analysis of mutations at the junction of the P4-P6 stacked helices. J. Mol. Biol. 301, (2000). 9. Caprara, M. G., Myers, C. A. & Lambowitz, A. M. Interaction of the Neurospora crassa mitochondrial tyrosyl-trna synthetase (CYT-18 protein) with the group I intron P4-P6 domain. Thermodynamic analysis and the role of metal ions. J. Mol. Biol. 308, (2001). 10. Myers, C. A., Kuhla, B., Cusack, S. & Lambowitz, A. M. trna-like recognition of group I introns by a tyrosyl-trna synthetase. Proc. Natl. Acad. Sci. USA 99, (2002). 11. Webb, A. E., Rose, M. A., Westhof, E. & Weeks, K. M. Protein-dependent transition states for ribonucleoprotein assembly. J. Mol. Biol. 309, (2001). 12. Chen, X., Mohr, G. & Lambowitz, A. M. The Neurospora crassa CYT-18 protein C-terminal RNA-binding domain helps stabilize interdomain tertiary interactions in group I introns. RNA 10, (2004). 13. Yaremchuk, A., Kriklivyi, I., Tukalo, M. & Cusack, S. Class I tyrosyl-trna synthetase has a class II mode of cognate trna recognition. EMBO J. 21, (2002). 14. Laskowski R. A., MacArthur, M. W., Moss, D. S., & Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst., 26, (1993). 18