Crystallization, data collection, and structure determination
This protocol is extracted from research article:
MMOD-induced structural changes of hydroxylase in soluble methane monooxygenase
Sci Adv, Oct 2, 2019; DOI: 10.1126/sciadv.aax0059

Purified MMOH and MMOD [in solution containing Hepes, 30 mM (pH 7.5); NaCl, 100 mM; and TCEP, 1 mM] were mixed in a 1:2 molar ratio, and the final concentration was adjusted to 10 mg/ml. Plate-shaped crystals were grown within a week at room temperature using the hanging drop vapor diffusion method with a mixture of 10% (w/v) PEG 8000 (polyethylene glycol, molecular weight 8000), 20% (v/v) ethylene glycol, 0.02 M 1,6-hexanediol, 0.02 M 1-butanol, 0.02 M 1,2-propanediol, 0.02 M 2-propanol, 0.02 M 1,4-butanediol, 0.02 M 1,3-propanediol, 0.89 M 1,3-butanediol, and 0.1 M MES/imidazole buffer (pH 6.5). After transferring to a cryoprotectant solution containing the precipitant and 35% (w/v) PEG 8000, crystals were flash-frozen in liquid nitrogen. The space group of the crystal was C2 with a = 179.8 Å, b = 125.8 Å, c = 126.4 Å, α = γ = 90°, and β = 102.9°. The dataset was collected at Advanced Photon Source on beamline Life Sciences Collaborative Access Team (LS-CAT) 21 ID-G. The 2.6-Å data were processed by HKL2000 (www.hkl-xray.com), and initial phases were calculated by molecular replacement (Phaser) (34) using M. trichosporium OB3b MMOH as the search model (PDB ID: 1MHY) (20). After molecular replacement, the electron density for MMOD was clearly visible. The MMOD model was subsequently manually built using COOT (35), and the refinement was performed using phenix.refine (36). Iron atoms and ligand restraints were generated using the program PHENIX (phenix.metal_coordination and phenix.ready_set) and applied during the refinement. No noncrystallographic symmetry restraints were applied during the refinement. The asymmetric unit contains two copies each of the MMOHα, MMOHβ, and MMOHγ subunits and MMOD full-length, and the Rwork and Rfree values of the final refined model were 17.8 and 22.6, respectively. Residues 1 to 11 (1 to 10) and 161 to 172 (159 to 171) of the MMOHα, residues 1 to 56 (1 to 56) of the MMOHβ, residues 1 to 4 (1 to 2) of the MMOHγ, and residues 1 to 11 (1 to 6) and 76 to 111 (75 to 111) of MMOD were disordered and invisible in the final model (parentheses indicate residues in the other MMOH protomer). Ramachandran analyses were performed by MolProbity (37), and the results were as follows: 95.4 (favored), 4.5 (allowed), and 0.1% (outlier).

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