rotein crystals were cryoprotected with reservoir solution containing 20% glycerol and frozen in liquid nitrogen. Data was collected using synchrotron radiation with an oscillation angle of 0.5u and 360 recorded images at 1 second exposure for each at the Diamond Light Source, UK. Data Processing and Structural Determination The X-ray diffraction data was indexed and integrated using iMosflm, and scaled using Scala in ccp4 suite. The crystal structure of LASV L N-terminal region was determined by molecular replacement using Phaser and the N-terminal domain of LCMV as a search model. Model building was completed in Coot and structure refinements were carried out using Phenix. Crystal Structure of the L endonuclease Domain The crystals of the LASV L N-terminal region belong to a space group of P43212 with cell dimensions a = 57.72, b = 57.72, c = 134.51, and a = b = c = 90u. The crystal structure was determined by molecular replacement using the LCMV endonuclease structure as the search model and refined to 1.72-A resolution with an Rfactor of 16.54% and the Rfree of 17.83%. The protein contains an N-terminal domain that consists of a four a-helices bundle and a Cterminal domain that composes of three antiparallel b-sheets and two helices . Between the two domains is a highly positively charged groove, which we speculate to be a RNA binding site, and a highly negatively charged cavity, which harbors two magnesium ions and is a potential active site of the endonuclease domain. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19645691 In Vitro Endonuclease MedChemExpress Relebactam Assays A single-stranded 16-nucleotide RNA fragment with a fluorescence 6-carboxyfluorescein label at its 59 end was synthesized and HPLC purified by Eurogentec, and further purified using a 20% polyacrylamide/8 M urea gel. The endonucleolytic activity of the wild-type L173 was determined using the FAM-RNA as substrate in a control experiment in which the RNA substrate in solution containing 20 mM Tris, pH 7.5, 0.3 M NaCl, 10% glycerol and RNase inhibitor was incubated with purified LASV L173 at 37uC for 20 minutes in varying combinations of EDTA and divalent metal ions. Metal ion preference was tested by incubation of FAM-RNA substrate and LASV L173 with 0.5 mM of MgCl2, MnCl2, CaCl2 and ZnCl2 at 37uC for 20 minutes. Endonucleolytic cleavage of the alanine substitution mutants E51A, E102A and D119A was performed by incubation of FAMRNA in a 1:10 ratio with the individual mutated version of the protein in the presence of 2.5 mM MgCl2 and RNase inhibitor at 37uC. Samples were taken after 0, 25, 40 and 90 minutes incubation time and stopped by the addition of EDTA pH 8.0 and 100% formamide. Samples were heated to 95uC for 5 minutes prior to separation on a 20% PAGE/8 M urea gel. Vertical gel electrophoresis was carried out at 45uC and under protection from light for 150 minutes. Immediately after electrophoresis, the gel was scanned at an absorbance of 500 nm using a Typhoon scanner and the intensity of the bands was quantified by the ImageJ software. The Potential Catalytic Residues of the Lassa L Endonuclease Domain Two Mg2+ ions, presumably picked up by the protein from the crystallization solution, are located at the presumed active site of the LASV endonuclease domain. Four water molecules and the side chain of D89 coordinate the first Mg2+, while three water molecules, the side chain of D89 and the main chain oxygen of C103 coordinate the second Mg2+. This metal binding feature is different from that of the known structures of the influenza
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