EC Number   |
|---|
  4.99.1.9 | purified recombinant His-tagged enzyme in apoform and with bound product coproheme, mixing of 9.5 mg/ml protein with or without bound ligand in solution with 16% w/v PEG 8000, 20% v/v glycerol, and 0.04 M KH2PO4 for the apo-enzyme crystals and with 0.1 M Bis-Tris, pH 5.5, 25% PEG 3350, 0.2 M MgCl2 for the complexed enzyme, 2 days, X-ray diffraction structure determination and analysis, molecular replacement using the PDB structure 2HK6 of BsCpfC enzyme as template |
| 4.99.1.9 | purified wild-type and mutant R45L enzymes in apo and in substrate coproporphyrin III-bound forms, apoprotein-LmCpfC is dissolved in 50 mM HEPES, pH 7.4, and mixed with 0.0.05-0.010 mM cpIII in 50 mM HEPES, pH 7.4, the crystallization solution contains 15-25% PEG MME 2000, 0.1 M Bis-Tris, pH 5.8-6.3, and 0.1-0.4 M calcium acetate, mixing of 150 nl each. The LmCpfC R45L mutant in complex with cpIII is crystallized in 18% w/v PEG 8000 and 20% glycerol with a 1:1000 dilution of apo wild-type germination solution containing crushed apo wild-type crystals and 5 mM phosphate buffer, one drop contains 150 nl of 0.35 mM cpIII-R45L protein, 200 nl of mother liquor, and 30 nl of 1:1000 seed solution, X-ray diffraction structure determination and analysis at 1.51 A and 2.64 A resolution, respectively, molecular replacement using the apo-LmCpfC structure (PDB ID 6RWV) and refined to Rfree values of 0.1816 (cpIII-LmCpfC wild-type) and 0.2112 (cpIII-LmCpfC R45L) as templates |
| 4.99.1.9 | structure in presence of iron. Only a single iron ion is found in the active site, coordinated in a square pyramidal fashion by two amino acid residues, His183 and Glu264, and three water molecules. This iron ion is not present in the structure of a His183Ala modified ferrochelatase. Insertion of a metal ion into protoporphyrin IX by ferrochelatase occurs from a metal binding site represented by His183 and Glu264 |
| 4.99.1.9 | the three-dimensional structure is determined at 1.9 A resolution by the method of multiple isomorphous replacement. The structural model contains 308 of the 310 amino acid residues of the protein and 198 solvent molecules |
