Supplementary Materials s5

Supplementary Materials

Figure Preparation

Figures were prepared using MOLSCIPT 1 through a graphical user interface (MOLDRAW) supplied by N. Strater (N. Strater, Leipzig, Germany, unpublished). All rendering was done with RASTER3D 2. Molecular surface representations have been calculated with MSMS 3. Electrostatic potentials were calculated with DELPHI 4 using standard parameters. Sequence logo representations of multiple sequence alignments were generated using the WebLogo server 5.

1. Kraulis, P. J. MOLSCRIPT: A Program to Produce Both Detailed and Schematic Plots of Protein Structures. J. Appl. Cryst. 24, 946-950 (1991). 2. Merritt, E. A. & Bacon, D. J. Raster3D:Photorealistic Molecular Graphics. Methods Enzym. 277, 505-524 (1997). 3. Sanner, M. F., Olson, A. J. & Spehner, J. C. Reduced surface: an efficient way to compute molecular surfaces. Biopolymers 38, 305-20 (1996). 4. Rocchia, W. et al. Rapid grid-based construction of the molecular surface and the use of induced surface charge to calculate reaction field energies: applications to the molecular systems and geometric objects. J. Comput. Chem. 23, 128-37 (2002). 5. Crooks, G. E., Hon, G., Chandonia, J. M. & Brenner, S. E. WebLogo: a sequence logo generator. Genome Res. 14, 1188-90 (2004). Supplementary Figures and Tables

S1: Example of experimental electron density quality. FOM-weighted experimental electron density map after solvent flipping contoured at 1.4 sigma shown together with the C-trace of the final refined model.

S2: Structural flexibility of the Trigger Factor. Superimposition of the two full-length Trigger Factor molecules in the crystallographic asymmetric unit. Molecule A is shown in blue, molecule B in red.

S3: Crystal contact sites of the full-length Trigger Factor. Trigger Factor is coloured by domains as in Fig. 1a. Positions of residues involved in crystal contacts are shown as light and dark spheres for the contacts with two distinct symmetry mates of the molecule shown.

S4: E.coli Trigger Factor associates with Haloarcula marismortui 50S and crosslinks to the ribosomal protein L23. Purified H. marismortui 50S was incubated either with Trigger Factor wild type (TF WT), a TF mutant deficient in ribosome binding (TF- AAA) or the TF mutant TF-V49C. Pelleted H. marismortui 50S together with associated chaperones was analysed by Coomassie stained SDS-PAGE. All TF variants, except TF-AAA, were associated with H. marismortui 50S subunit and were detected in the ribosomal pellet (P). Upon UV irradiation TF-V49C labelled with BPIA crosslinked to a ribosomal component (lane 9) identified as L23 by mass spectrometry. TF variants were added in a physiological twofold molar excess over ribosomes and were therefore also detected in the supernatant (S).

S5: Sequence conservation of L23 and ribosomal RNA at the TF binding site. a. Sequence-logo (http://weblogo.berkeley.edu/) representation of aligned sequences of 24 archaeal and 160 bacterial L23 proteins. The H. marismortui and E.coli L23 sequences are given in the inset. b. H. marismortui and E.coli ribosomal RNA sequences around the TF interacting A1501 in secondary structure representation. In a. and b. residues in contact with TF are indicated by asterisks.

Table 1: Data collection and refinement statistics. a. Structure determination of the full-length Trigger Factor. b. Structure determination of the Trigger Factor fragment TF 1-144 in complex with the H. marismortui 50S ribosomal subunit.