Computational protocol: Structure and Haem-Distal Site Plasticity in Methanosarcina acetivorans Protoglobin

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[…] The cyanide derivative of MaPgb*(III) and of its mutants was crystallized by vapor diffusion techniques (protein concentrations ∼45 mg/ml) under conditions matching those for the ligand-free MaPgb*(III) . In particular, crystals of MaPgb*(III)-cyanide were grown by equilibrating the protein solution against a precipitant solution containing 30% PEG 4000, 0.2 M Li2SO4, 0.1 M Na Hepes (pH 7.0–7.5), 0.02 M potassium ferricyanide, and 0.01 M KCN (at 4°C). Crystals belong to the monoclinic space groups P21 (two MaPgb* molecules in the asymmetric unit) or C2 (one MaPgb* molecule in the asymmetric unit). The best crystals diffracted to 1.6 Å resolution using synchrotron radiation (ESRF, Grenoble, France). Crystals of the cyanide-bound ferric Trp(60)B9Ala, Tyr(61)B10Ala, and Leu(142)G4Ala mutants were obtained by equilibrating the protein solutions (containing 0.005 M KCN) against 20–25% PEG 4000, 10% isopropanol, 0.1 M Na Hepes (pH 7.0–7.5), 0.02 M potassium ferricyanide, and 0.01 M KCN (at 4°C). All crystals belong to the primitive monoclinic P21 space group (two MaPgb* molecules in the asymmetric unit) and diffracted to high resolution (in the 1.5 Å–1.7 Å range) using synchrotron radiation (ESRF, Grenoble, France). Crystallization conditions similar to those described above for the MaPgb*(III) mutants produced also crystals belonging to the monoclinic C2 space group (three MaPgb* molecules in the asymmetric unit) for the Phe(93)E11Leu, and Ile(149)G11Phe mutants, which diffracted up to 2.0 Å and 1.5 Å resolution, respectively, using synchrotron radiation (ESRF, Grenoble, France).The azide derivative of MaPgb*(III) was prepared by adding to the MaPgb*(III) solution (about 43 mg/ml concentration) 0.01 M potassium ferricyanide and 0.1 M Na azide (NaN3). After 1 h of incubation, the protein-azide complexes were equilibrated against precipitant solutions containing 20–30% w/v PEG 4000, 0.2 M Li2SO4 or 10% isopropanol, 0.1 M Na Hepes (pH 7.0–7.5), at 4°C. The best MaPgb*(III)-azide crystals were grown either at 30% w/v PEG 4000, 0.2 M Li2SO4, 0.1 M Na Hepes (pH 7.5), or at 20% w/v PEG 4000, 10% isopropanol, 0.1 M Na Hepes (pH 7.0). In the first case, the crystals belong to the monoclinic C2 space group (two MaPgb* molecules in the asymmetric unit) and diffracted up to 1.8 Å, using synchrotron radiation (ESRF, Grenoble, France). In the second case, the crystals belong to the monoclinic P21 space group (two MaPgb* molecules in the asymmetric unit); one of these was used for Xenon binding experiments. To promote Xenon diffusion within the protein matrix, the MaPgb*(III)-azide crystal was exposed to 10 bar Xenon for 5 min in a high-pressure chamber (Xcell, Oxford Cryo-system, UK), and rapidly transferred to liquid nitrogen. X-ray diffraction data up to 2.3 Å resolution were collected using synchrotron radiation (ESRF, Grenoble, France). An identical Xenon-binding procedure was also applied to the MaPgb*(III)-cyanide crystals (C2 crystal form) to produce the MaPgb*(III)-cyanide-Xenon complex.The imidazole- and nicotinamide-bound MaPgb*(III) complexes were prepared by adding to the MaPgb*(III) solution (about 20 mg/ml concentration) 0.01 M potassium ferricyanide and 0.04 M either imidazole or nicotinamide. After 1 h of incubation, the MaPgb*(III)-ligand complexes were equilibrated against a precipitant solution containing 0.25–0.5 M monobasic ammonium phosphate or against 15–25% w/v PEG 4000, 10% v/v 2-propanol, and 0.1 M Na Hepes (pH 7.0–7.5), at 4°C. The best MaPgb*(III)-imidazole crystals grew in 0.4 M monobasic ammonium phosphate, matching the precipitant solution condition successfully used for the crystallization of the MaPgb*(II)-O2 complex . The crystals belong to the monoclinic C2 space group (two MaPgb* molecules in the asymmetric unit) and diffracted up to 1.38 Å resolution using synchrotron radiation (ESRF, Grenoble, France). The best MaPgb(III)-nicotinamide crystals grew in 18% w/v PEG 4000, 10% v/v isopropanol, and 0.1 M Na Hepes (pH 7.5). They belong to the monoclinic P21 space group (two MaPgb* molecules in the asymmetric unit) and diffracted up to 1.9 Å resolution using synchrotron radiation (ESRF, Grenoble, France). Statistics for each data collection are reported in details in and .All X-ray diffraction data were integrated and reduced using MOSFLM and SCALA , ; structure determination was achieved by molecular replacement methods with the program PHASER , using the MaPgb*(II)-O2 structure as the search model (PDB accession code 2VEB) . Crystallographic refinement was performed using the program REFMAC , the program COOT having been used for model building/inspection. The relevant refinement statistics are reported in and . The program Procheck was used to assess the stereochemical quality of the protein structures.Atomic coordinates and structure factors have been deposited with PDB accession codes 3ZJN (MaPgb*(III)-cyanide complex), 3ZJR (MaPgb*(III)-cyanide-Xenon complex), 3ZJO (MaPgb*(III)-azide complex), 3ZJS (MaPgb*(III)-azide-Xenon complex), 3ZJP (MaPgb*(III)-imidazole complex), 3ZJQ (MaPgb*(III)-nicotinamide complex), 3ZJH (Trp(60)B9Ala-cyanide complex), 3ZJI (Tyr(61)B10Ala-cyanide complex), 3ZJJ (Phe(93)E11Leu-cyanide complex), 3ZJL (Leu(142)G4Ala-cyanide complex), and 3ZJM (Ile(149)G11Phe-cyanide complex). […]

Pipeline specifications

Software tools Coot, PROCHECK
Applications Small-angle scattering, Protein structure analysis
Organisms Methanosarcina acetivorans C2A, Methanosarcina acetivorans
Chemicals Hydrogen, Niacinamide