Supplementary Information
Methods
Purification of E. coli 70S RNCs and SRP
For the generation of RNCs we used the in vitro Rapid Translation System RTS 100 E. coli HY Kit from Roche. Ribosomes were programmed with truncated mRNA encoding the 102 N-terminal amino acid fragment of the E. coli membrane protein FtsQ. RNCs were then purified using an additional N-terminal His-tag1. The DNA encoding the FtsQ-fragment was amplified by PCR using genomic E. coli DNA as template and an extended forward primer for introducing a T7 promotor site followed by a high initiation efficiency 5´-untranslated region plus hexa-histidine and HA-tags. The reverse primer defined the length of the nascent chain to the first 102 amino acids of FtsQ. mRNA was synthesized using the T7-MEGAshortscript Kit from Ambion with the DNA-fragments as templates.
For translation two 400l reactions were incubated at 30°C for 30 min. Translation was stopped by transferring the reaction to 4°C. Each reaction was spun through 300l of a high salt sucrose cushion (50 mM Tris/HCl (pH 7.2), 500 mM KOAc, 25 mM Mg(OAc)2, 5 mM 2-mercaptoethanol, 0.75 M sucrose , 0.1% Nikkol, 500 g ml-1 chloramphenicol, and 0.1% pill/ml [1 pill complete protease mix /ml H2O, Roche Diagnostics GmbH) at 50,000g for 178 min in a TLA 120.2 rotor. Each pellet was resuspended in 1 ml ice cold 250 buffer (50 mM Tris/HCl (pH 7.2), 250 mM KOAc, 25 mM Mg(OAc)2, 5 mM 2-mercaptoethanol, 250 mM sucrose, 0.1% Nikkol, 500 g ml-1chloramphenicol, 0.2 U ml-1 RNAsin, 0.1% pill/ml, transferred on 750 l of Talon Metal Affinity Resin (BD) and incubated for 20 min at RT. The resin was washed with 10 ml ice cold 250 buffer, and 3 ml 500 buffer (250 buffer but 500 mM KOAc). RNCs were eluted with 2.5 ml 250 buffer supplemented with 100 mM imidazol (pH 7.0). The eluted RNCs were spun through 200 l of a high salt sucrose cushion at 50,000g for 137 min in a TLA 100.4 rotor and the resulting pellet was resuspended slowly in grid buffer (20 mM Hepes (pH 7.2), 50 mM KOAc, 6 mM Mg(OAc)2, 1 mM DTT, 500 g ml-1 chloramphenicol, 0.05% Nikkol, 0.5% pill ml-1 and 125 mM sucrose), flash frozen and stored at –80°C. The yield of isolated 70S RNCs was typically 2 OD260 with a concentration of 30 OD ml-1. Recombinant E. coli SRP was over-expressed and affinity purified.
Reconstitution of E. coli 70S RNC-SRP complexes
RNC-SRP complexes were reconstituted by incubating 3 pmol RNCs with 15 pmol SRP for 20 min at RT followed by 10 min at 37°C in a final volume of 20 l of buffer D (20 mM Hepes-KOH (pH 7.4), 150 mM KOAc, 10 mM Mg(OAc)2, 4 mM 2-mercaptoethanol, 5 mM spermidin, 0.05 mM spermin). Binding was checked by sucrose density gradient centrifugation followed by SDS-PAGE and Western Blotting.
Electron microscopy, image processing and models
Samples were applied to carbon-coated holey grids2. Micrographs were recorded under low-dose conditions on a Tecnai F30 field emission gun electron microscope at 300 kV and scanned on a Heidelberg drum scanner with a pixel size of 1.23 Å on the object scale. The contrast transfer function was determined with CTFFIND and SPIDER3. After automated particle picking with Signature followed by visual inspection the data were processed with the SPIDER software package and classified into several subsets according to ribosomal and ligand conformations4. 26,000 particles from the E. coli RNC-SRP dataset were used for the final CTF-corrected reconstruction with the resolution of 9.4 Å (5.6 Å) based on the Fourier shell correlation with a cutoff value of 0.5 (3). 50,000 particles with the P-site tRNA in the programmed ribosome and without a ligand were back-projected resulting in the signal sequence containing RNC at a resolution of 9.1 Å (5.5 Å). The resolution of the previously published mammalian SRP-RNC complex1 was improved by adding more particles to the dataset and extensive sorting, resulting in the final set of 23,000 particles and a reconstruction at a resolution of 8.7 Å (5.1 Å).
Densities for the large and small ribosomal subunit, the P-site tRNA, the E. coli SRP and mammalian SRP were isolated using binary masks. Amplitude correction was done by Fourier filtering using B-factors. A lower contour level of the ligand densities for surface representation was applied. This indicates that ligand densities are partially flexible or still under-represented because of incomplete removal of ligand-free ribosomal particles from the final particle subset. For the same reasons the resolution of the ribosome-bound ligands such as SRP is somewhat lower than the resolution of the ribosome itself. Nevertheless, in the domains we used for flexible model adjustment the resolution of the ligands allowed the recognition of α-helical secondary structure.
Docking of X-ray structures and molecular models was performed using the programs O5 and Situs6. The mammalian SRP structure was docked using O5 into the density with resolved -helices and the flexible N-terminal part of M-domain was adjusted accordingly. The C-terminus of mammalian SRP54 was modeled using protein threading. Several possible models were created and the model based on E. coli arsenite translocating ATPase (1ihu_A) as a template was fitting well into the density and was docked with only a minor adjustment of its most C-terminal helix.
The crystal structure of E. coli 50S subunit (2AW4)7 was docked using Situs6. The E. coli SRP (1DUL)8 and S. sulfatoricus SRP (1QZW)9 were docked using O and the flexible N-terminal part was remodeled into the density. For visualization B-factors were applied (30/Ų for the ribosome, 15/Ų for SRPs). Figures were prepared using Chimera10.
1.Halic, M. et al. Structure of the signal recognition particle interacting with the elongation-arrested ribosome. Nature427, 808-14 (2004).
2.Wagenknecht, T., Grassucci, R. & Frank, J. Electron microscopy and computer image averaging of ice-embedded large ribosomal subunits from Escherichia coli. J Mol Biol199, 137-47. (1988).
3.Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J Struct Biol116, 190-9 (1996).
4.Penczek, P. A., Frank, J. & Spahn, C. M. A method of focused classification, based on the bootstrap 3D variance analysis, and its application to EF-G-dependent translocation. J Struct Biol154, 184-94 (2006).
5.Jones, T. A., Zhou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. AA47, 110-119 (1991).
6.Wriggers, W., Milligan, R. A. & McCammon, J. A. Situs: A package for docking crystal structures into low-resolution maps from electron microscopy. J Struct Biol125, 185-95 (1999).
7.Schuwirth, B. S. et al. Structures of the bacterial ribosome at 3.5 A resolution. Science310, 827-34 (2005).
8.Batey, R. T., Rambo, R. P., Lucast, L., Rha, B. & Doudna, J. A. Crystal structure of the ribonucleoprotein core of the signal recognition particle. Science287, 1232-9. (2000).
9.Rosendal, K. R., Wild, K., Montoya, G. & Sinning, I. Crystal structure of the complete core of archaeal signal recognition particle and implications for interdomain communication. Proc Natl Acad Sci U S A100, 14701-6 (2003).
10.Pettersen, E. F. et al. UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem25, 1605-12 (2004).
Table 1 Statistics of cryo-EM and single particle reconstruction
70S RNC / 70S RNC-SRP / 80S RNC-SRPNumber of particles / 50,000 / 26,000 / 23,000
Pixel size / 1.23 Å / 1.23 Å / 1.23 Å
Resolution (FSC 0.5) / 9.1 Å / 9.4 Å / 8.7 Å
Resolution (FSC 3 / 5.5 Å / 5.6 Å / 5.1 Å
Ligand resolution (0.5) / - / 9.5-12 Å / 9.5-11.5 Å
B-factors of ribosome / 30/Ų / 30/Ų / 30/Ų
B-factors of ligands / - / 15/Ų / 15/Ų