480 Nucleic Acids Research, 2005, Vol. 33, No. 2 [e.g. lysine instead of arginine, as in (13)] the host strain was co-transformed with pUBS520, a plasmid carrying the gene for tRNAARG AGA=AGG (14). For MAD phasing, selenomethionine- MjaL1 was produced by essentially the same procedure but with E.coli strain B834(DE3) and minimal media containing selenomethionine (15). Cells were suspended in 100 mM Tris–HCl buffer (pH 7.5) (25 C), 0.8 M NaCl, 100 mM MgCl2, 0.2 mM EDTA, 5 mM b-mercaptoethanol, 0.1 mM PMSF and disrupted by brief sonication. The cell debris and the ribosomes were removed by centrifugation. The main con- taminating proteins in the supernatant were then precipitated by heating for 10 min at 65 C and removed by centrifugation. The supernatant was diluted with 50 mM sodium acetate (pH 5.5) to adjust the concentration of NaCl to 0.15 M and the protein was puriﬁed by cation-exchange (CM-Sepharose) chromatography. A linear gradient from 0.15 M to 0.8 M NaCl was used for elution. The protein preparation was dialyzed into 10 mM sodium cacodylate buffer, pH 6.0, containing 100 mM NaCl. Filter-binding assay Uniformly 32P-labeled RNA fragments were synthesized in vitro from templates linearized by SmaI in the presence of [a-32P]UTP (800 Ci/mmol; New England Nuclear Corp.) using the MAXIscript T7 Kit (Ambion Inc., Austin, TX). Unincorporated nucleotides were removed with QIAquick Nucleotide Removal Kit (QIAGEN Inc.). The purity and integrity of the transcripts were conﬁrmed by electrophoresis on 12.5% polyacrylamide gels containing 8 M urea. The afﬁn- ity of MjaL1 to RNA-binding fragments was determined by nitrocellulose ﬁlter-binding assay as described previously (10). The binding buffer contained 50 mM Tris–HCl, pH 7.6, 20 mM MgCl2, 350 mM KCl, 1 mM b-mercaptoethanol and 0.04% BSA. Crystallization The RNA fragment and the protein were mixed in equimolar amounts (3 mg/ml for RNA and 4.5 mg/ml for MjaL1). Crystals were grown by the vapor diffusion method at 22 C. Hanging drops were made by mixing MjaL1–mRNA complex with 3% PEG 10 K, 300 mM KCl, 50 mM sodium cacodylate buffer, pH 6.0 and 6% 2-methyl-2,4-pentandiol, 120 mM KCl in a 6:1:1 volume ratio, respectively, and the well solution was made from 30% PEG 10 K in 100 mM sodium cacodylate buffer, pH 6.0. Crystals appear within ﬁve days. Before freezing in liquid nitrogen, the crystals were transferred to the solution composed of 21% butane-2,3-diol, 1.5% PEG 10 K, 60 mM KCl, 50 mM sodium cacodylate, pH 6.0. Data collection and structure determination Data were collected from a single SeMet crystal of MjaL1– mRNA at the MPG/GBF beamline BW6, DESY (Hamburg, Germany) using a MAR CCD detector and were processed and merged with the XDS program suite (16). The crystal structure of the regulatory complex MjaL1–mRNA was initially solved by the MAD method in space group I222. An electron density map of a workable quality was obtained and allowed us to build models of both mRNA and L1 molecules within the complex. However, we could not reﬁne this structure to an R-free value less than 40%. Detailed analysis of experimental data shows that crystals of the L1–mRNA complex are pseudohemihed- rally twinned and belong to space group C2 with unit cell parameters a = 212.3 s, b = 68.9 s, c = 115.9 s and b = 123.0 , and two monomers in the asymmetric unit. Because for these crystals a · cosb is almost equal to c, the cell can be indexed as I222 with a = 68.9, b = 116.1 and c = 178.5 s, where orthorhombic a-axes are parallel to a* axes of the monoclinic reciprocal unit. The cumulative intensity distribution calculated with TRUNCATE of the CCP4 pro- gram suite (17) indicated pseudohemihedral twinning in space group C2. The twin law h + 2l, k, l describes a real space rotation about an axis perpendicular to the crystallographic 2-fold. The twin fraction was estimated to be 0.35 by the Britton plot (18). The location of the complex in the correct unit cell was done by the molecular replacement method using the obtained structure as a model. The molecular replacement yielded an unambiguous solution with a correlation coefﬁcient of 70.7% and R-factor of 41.2%. Unfortunately, the electron density map calculated with detwinned structure-factor ampli- tudes and model phases was of lower quality than with twinned data. Therefore, we used twinned data for the model building and reﬁnement. The ﬁnal model, reﬁned to an R-factor of 27.5% and an R-free of 31.4% at 3.4 s resolution, includes 214 amino acid residues and 49 nt. Data and reﬁnement statis- tics are summarized in Table 2. Determination of heavy atom positions, initial phasing, density modiﬁcation and reﬁnement were executed using CNS (19). The map interpretation and model building were performed with O (20). NCS restraints were used during the early stages of reﬁnement, but the two molecules in the asymmetric unit were ﬁnally reﬁned separately. The structural data and the coordinates have been deposited in the Protein Data Bank (accession code 1U63). Table 2. Data collection and refinement statistics Crystallographic dataa Space group Unit-cell parameters (s, ) Wavelength (s) Resolution (s) Number of reflections Number of unique reflections Completeness (%) Averaged redundancy I/si(I) Rsym(I) (%)b Refinement statistics Resolution range (s) Reflections R-factor (%) Free R-factor (%) r.m.s. deviation Bond lengths (s) Bond angles ( ) Improper angles ( ) Mean B value (overall, s2) C2 a = 212.3, b = 68.9, c = 115.9, a = 90 , b = 123.0 , g = 90 1.05 30–3.40 (3.61–3.40) 33 549 (5012) 17 645 (2718) 89.4 (83.9) 1.9 (1.84) 9.68 (2.79) 6.2 (31.8) 8–3.4 16 761 (2066) 27.5 (38.5) 31.4 (43.0) 0.0104 1.87561 4.56 70.4 aValues in parenthesis are statistics for the highest resolution shell. bRsym = P j I h Ii j PhIi, where I is the measured intensity of each reflection and hIi is the intensity averaged from several observations of symmetry-related reflections.