NuscriptsSummary MethodA fusion protein (SRP54) consisting of Sulfolobus solfataricus SRP54 and yeast dipeptidyl aminopeptidase B signal sequence was made in E. coli. Crystal structure of SRP54 was Ganglioside GD3 (disodium salt) Technical Information determined by the MAD system utilizing a methylmercury derivative of a mutant (N177C).MethodsPlasmid for protein expression The sequence encoding Sulfolobus solfataricus SRP54 residues 232 was amplified by PCR from genomic DNA and Acrylate Inhibitors MedChemExpress cloned in to the NcoIXhoI website of pET15b. 4 overlapping oligonucleotides encoding the signal anchor sequence from S. cerevisiae dipeptidylaminopeptidase B (KLIRVGIILVLLIWGTVLLLKSIPHH) plus a pentahistidine tag, were cloned into the BamHI web-site in such a way that a BamHI site is retained only on the 5 end. This signal sequence was applied for any cryoEM study of your SRPribosomenascent chain complex12. A pair of oligonucleotides encoding many linker sequences was cloned between the XhoI and BamHI internet sites (Table S1). The 11 residue linker sequence was (ARSGSGSGSGS). A single cysteine mutant N177C was generated by a PCR primarily based mutagenesis. Protein expression and purification Rosetta (DE3) pLysS cells (Novagen) harbouring pET15SRP54 or pET15SRP54(N177C) have been grown in 2xTY media with 50g/ml Ampicillin and 25g/ml Chloramphenicol, and protein expression was induced at an OD600 of 0.7 with 1 mM IPTG for 3 hr at 25 . Harvested cells had been suspended in 20 mM Hepes pH 7.four, 1 M NaCl, ten glycerol and EDTAfree protease inhibitor cocktail (Roche) and lysed by sonication. The clarified lysate was applied to a NiNTA agarose (QIAGEN) column equilibrated in 20 mM Hepes pH 7.four, 500 mM NaCl, 20 mM imidazole and eluted with a linear gradient to 20 mM Hepes pH 7.four, 500 mM NaCl, 320 mM imidazole. The peak fractions had been applied to a hydroxyapatite (BioRad) column equilibrated in 20 mM TrisHCl pH 7.four, 200 mM NaCl and eluted having a linear gradient of 012 ammonium sulfate in the similar buffer. The pooled fractions have been dialysed against 20 mM TrisHCl pH 7.4, 200 mM NaCl and applied to a heparinSepharose column (Amersham Biosciences) equilibrated together with the similar buffer. The protein was eluted with a linear gradient of 200 mM1 M NaCl in 20 mM TrisHCl pH 7.4. Peak fractions have been pooled and dialysed against 20 mM Tris HCl pH 7.4, one hundred mM NaCl. At this salt concentration, dimers remained soluble whereas all greater oligomers precipitated. Crystallization SRP54 dimer crystals were grown by hanging drop vapour diffusion at 295 K, by mixing equal volumes of protein (18 mg.ml1) and reservoir answer containing 57 PEG 4000,Nature. Author manuscript; obtainable in PMC 2010 November 27.Janda et al.Page100 mM BisTris pH 5.5, one hundred mM NaCl, 2 Polypropylene glycol P400, 550 mM Mg(OAc)two. Single crystals were obtained by streak seeding from current SRP54 crystals. Purified SRP54(N177C) was prereacted with 0.5 mM methylmercury nitrate and crystallised beneath exactly the same situation. The native and derivative crystals grew within the space group P41212 and appeared within 57 days. The crystals were equilibrated with 25 PEG 4000, 15 Glycerol, 100 mM BisTris pH 5.five, one hundred mM NaCl and flash frozen in liquid nitrogen. Structure determination The native dataset and threewavelength MAD datasets (peak, inflection and remote) of the methylmercury derivative were collected at 100 K on beamline ID141 and ID144 at the European Synchrotron Radiation Facility in Grenoble, France. The information have been processed with MOSFLM/SCALA/TRUNCATE25,26. The single methylmercury web site was determined in AutoS.
