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EC Number Crystallization (Commentary)
Show all pathways known for 2.7.4.16Display the word mapDisplay the reaction diagram Show all sequences 2.7.4.16purified recombinant enzyme in complex with nonhydrolyzable AMP-PCP and TMP, or with the reaction products ADP and TPP, ThiL is incubated with 4 mM AMP-PCP or ATP and 4 mM MgCl2 on ice for 1 h prior to crystallization experiments, optimized conditions for the ThiL binary complexes include 100 mM HEPES, pH 7.4, 6-11% isopropyl alcohol, 200 mM NaCl, and 4-6% 2-methyl-2,4-pentanediol, 100 mM CaCl2 is used as an additive in a 1:9 ratio with the reservoir solution, TMP is soaked into the co-crystallized ThiL and AMP-PCP crystals by transferring the crystals to a solution containing the mother liquor and 20 mM TMP and allowing the crystals to soak for 5 min before being frozen. To obtain ThiL ADP-TPP crystals, ThiL is incubated on ice with 4 mM ADP, 4 mM TPP, and 4 mM MgCl2, optimized conditions for the product complex include 100 mM imidazole, pH 8.0, 7-10% PEG 8000, and 100-250 mM Ca(C2H3O2)2, 3-7 days, cryoprotection by 15% ethylene glycol, X-ray diffraction structure determination and analysis at 1.5-2.6 A resolution
Show all pathways known for 2.7.4.16Display the word mapDisplay the reaction diagram Show all sequences 2.7.4.16sitting drop method, crystal structures of thiamine monophosphate kinase in complex with a pair of substrates TMP and a non-hydrolyzable adenosine triphosphate analog, and in complex with a pair of products TPP and adenosine diphosphate
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