Six genes, Lsm1, Lsm2, Lsm3, Lsm5, Lsm6, and Lsm7, were amplified from the

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1 Supplementary information, Data S1 Methods Clones and protein preparation Six genes, Lsm1, Lsm2, Lsm3, Lsm5, Lsm6, and Lsm7, were amplified from the Saccharomyces cerevisiae genomic DNA by polymerase chain reaction (PCR). Open reading frames of Lsm4 was chemically synthesized to optimize codon usage for more efficient bacterial expression. All seven genes Lsm1 through Lsm7 were individually cloned into pqlink vector using BamHI and NotI cutting sites, with Lsm4 tagged by 7xHis at its N-terminus and Lsm6 by GST at its N-terminus. Then the seven vectors were combined step-by-step by ligation-independent pathway [1]. The pqlink vector with all seven Lsm genes was transformed into E. coli. Cells were grown to an optical cell density at 600 nm (OD 600 ) of at 37 C, and the expression of recombinant proteins was induced by 0.2 mm isopropyl-β-d-thiogalactopyranoside (IPTG) at 18 C overnight. Cells were harvested by centrifugation, resuspended in 25 mm Tris-HCl (ph 8.0), 150 mm NaCl, and disrupted by sonication. The recombinant proteins were purified through Ni 2+ -nitrilotriacetate affinity resin (Ni-NTA, Qiagen) and Glutathione Sepharose 4B column (GE Healthcare). TEV protease was applied to remove the 7xHis and GST tags before the Lsm protein complex was loaded into an anion exchange column (SOURCE-15Q, GE Healthcare). To prepare for the crystallization trials, the proteins were applied to gel filtration chromatography (Superdex /300 GL, GE Healthcare) in the buffer containing 15 mm HEPES, 6

2 ph 7.0, 200 mm NaCl. The peak fractions were collected and concentrated to 20 mg/ml for crystallization. The identity of the heptameric Lsm1-7 complex was confirmed by mass spectrometry. For production of selenomethionine (Se-Met) labeled protein, E. coli cells were grown in SelenoMet TM base medium plus nutrient mix (Molecular Dimension Limited). Selenomethionine was added into the medium before the expression of recombinant proteins was induced by 0.2 mm IPTG at 18 C overnight. The protein purification protocol was the same with unlabeled protein except that 1mM Tris [2-carboxyethyl] phosphine (TCEP) was included during the purification steps. Protein engineering for expression and crystallization We successfully generated the heptameric Lsm1-7 protein complex using the pqlink vector by co-expressing all seven components simultaneously. Because the expression level of Lsm4 was the lowest among the seven components, the 7xHis tag was placed at the N-terminus of Lsm4. Unfortunately, the full-length Lsm4 protein was degraded severely during protein purification. Sequence alignment among Lsm4 proteins from different species revealed the non-conserved nature of the extended, Asn-rich C-terminal half of Lsm4 from S. cerevisiae. The C-terminally truncated Lsm4 (residues 1-93) was over-expressed in stable form. Full-length Lsm1 also suffered the same problem of degradation. On the basis of the result of limited proteolysis, the N-terminal residues 1-29 of Lsm1 were truncated. The heptameric Lsm1-7 complex was successfully purified and exhibited excellent solution behavior. Rigorous 7

3 crystallization trials, involving screening more than 10,000 conditions, failed to generate any crystals. We suspected that the inability to crystallize the Lsm complex was due to heterogeneity the presence of a small proportion of non-heptameric Lsm complex involving Lsm4. To help improve homogeneity of the Lsm1-7 complex, the GST tag was fused to Lsm3 or Lsm6, as these two subunits were predicted to be located at opposing sides of the Lsm complex. With two tags, the Lsm1-7 complex exhibited markedly improved homogeneity, as evidenced by a single symmetric peak on anion exchange chromatography (SOURCE 15Q). Unfortunately, this Lsm1-7 complex still defied all efforts of crystallization. Reasoning that surface-exposed Cys residues may be prone to oxidation and thus hinder crystallization, we replaced all three Cys residues in the Lsm1-7 complex by Ser: C45S in Lsm2 and C37S/C63S in Lsm3. Finally, we were able to generate crystals of the Lsm1-7 complex (Lsm , Lsm , C45S in Lsm2, C37S/C63S in Lsm3). These crystals diffracted X-rays beyond 3 Å. Crystallization Extensive crystallization screens were performed at both 18 C and 4 C. Crystals of tetragonal rod appeared in two days and grew to full size in one week using the hanging-drop vapor-diffusion method. Crystals of both native and Se-Met labeled protein grew in 100 mm MES (ph 6.5), 25% PEG600 and 20 mm CaCl 2. Crystals of the native and Se-Met Lsm1-7 complex are in the space groups P2 1 and P1, respectively. Crystals were harvested and soaked in immersion oil types A (Cargille) 8

4 before flash-frozen in a cold nitrogen stream at 100 K. Data collection and structural determination All data sets were collected at the SSRF beamline BL17U and processed with the HKL2000 packages [2]. Further processing was carried out with programs from the CCP4 suite [3]. Data collection and structure refinement statistics are summarized in Supplementary Information Table 1. All crystals suffered the problem of twinning. Microbeam was applied for data collection. The Lsm1-7 structure was determined by molecular replacement combined with single anomalous diffraction (SAD) using the P1 datasets collected on the Se-Met-derived crystals. The molecular replacement was carried out by PHASER [4] using atomic coordinates of the heptameric Lsm protein (PDB code: 1I81) as the initial search model and two complexes were identified in each asymmetry unit. Then, using this partial model as input, the selenium positions were deterimned and phases were recalculated using the PHASER SAD experimental phasing module. With the improved map, cross-crystal averaging combined with solvent flattening, histogram matching and 2-fold NCS averaging was performed with the P2 1 dataset in DMMulti [5]. The atomic model was further rebuilt with COOT [6] and refined with PHENIX [7]. The sequence docking was aided by selenium anomalous signals in the P1 space group. Biolayer Interferometry(BLI) 9

5 Biolayer Interferometry(BLI)was carried out on a Fortebio Octet system according to the user guide. The dissociation rate (Kd) and association rate (Ka) were calculated. 5 -Biotin-labeled-RNA was purchased from TAKARA. Streptavidin biosensors (ForteBio Inc.) were hydrated for 10 minutes prior to the experiment in the buffer containing 15 mm HEPES (ph 7.0), 100 mm KCl, and 5 mm MgCl 2. Biotin-labeled RNA samples were diluted in the same buffer to 500 nm. After loading, the sensors were quenched with 5 μm biocytin and washed with the RNA loading buffer. After loading and quenching, the sensors with fixed biotin-labeled RNA were applied to measure binding affinity of different protein samples. The protein samples were diluted to different concentrations in the buffer containing 15 mm HEPES (ph 7.0), 100 mm KCl, 5 mm MgCl 2, 5 μg/ml BSA and 0.05% Tween-20, which is the same for the baseline buffer and the dissociation buffer. The data were fitted to a 1:1 binding mode. The binding affinity (K D ) values and standard deviations were generated by BLItz Pro software analysis. 1 Scheich C, Kummel D, Soumailakakis D, Heinemann U, Bussow K. Vectors for co-expression of an unrestricted number of proteins. Nucleic Acids Res 2007; 35:e43. 2 Otwinowski Z, Minor W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 1997; 276: Collaborative Computational Project N. The CCP4 suite: programs for protein crystallography. Acta Crystallogr 1994; D50: McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ. Phaser crystallographic software. J Appl Crystallogr 2007; 40: Cowtan K. DM: An automated procedure for phase improvement by density modification. Joint CCP4 and ESF-EACBM.. Newsletter on Protein Crystallography 1994; 31: Emsley P, Cowtan K. Coot: model-building tools for molecular graphics. Acta 10

6 Crystallogr D Biol Crystallogr 2004; 60: Adams PD, Grosse-Kunstleve RW, Hung LW et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr D Biol Crystallogr 2002; 58: