Computational protocol: Structural Bases for the Regulation of CO Binding in the Archaeal Protoglobin from Methanosarcina acetivorans

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Protocol publication

[…] The cyanide derivative of the ferric Phe(93)E11Tyr and Phe(145)G8Trp MaPgb* mutants (Phe(93)E11Tyr(III)-cyanide and Phe(145)G8Trp(III)-cyanide, respectively) was crystallized by vapor diffusion techniques (protein concentration ~2.6 mM) under conditions matching those for the ligand-free ferric protein[]. The mutant protein solutions were equilibrated against a precipitant solution containing 10% (v/v) isopropanol, 20% (v/v) PEG 4000, 0.1 M Na-Hepes (pH 7.5), 0.02 M potassium ferricyanide, and 0.01 M KCN at 277 K. The crystals diffracted to 1.6 Å and 2.2 Å resolution for the Phe(93)E11Tyr(III)-cyanide and Phe(145)G8Trp(III)-cyanide mutants, respectively (at 100 K), using synchrotron radiation (ESRF, Grenoble, France); both crystals belong to the primitive monoclinic P21 space group (). All collected data were reduced and scaled using the programs MOSFLM and SCALA, respectively, [,] and were phased by molecular replacement methods using the program PHASER []; the structure of ferrous oxygenated MaPgb* (MaPgb*(II)-O2) was used as the starting model (PDB accession code 2VEB) []. The crystallographic refinement was performed using the program REFMAC [], and the program COOT [] was used for model building/inspection. The relevant refinement statistics are reported in . The program Procheck [] was used to assess the stereochemical quality of the protein structures. Atomic coordinates and structure factors for Phe(93)E11Tyr(III)-cyanide and Phe(145)G8Trp(III)-cyanide have been deposited using the PDB accession codes 3ZOL and 3ZOM, respectively. […]

Pipeline specifications

Software tools iMosflm, CCP4, Coot, PROCHECK
Applications Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma, Methanosarcina acetivorans
Chemicals Heme