Computational protocol: The mechanism of catalysis by type-II NADH:quinone oxidoreductases

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[…] NDH-2 variants were crystallized by hanging-drop vapor diffusion at 18 °C using drops containing 0.5 μL of 30 mg mL−1 protein + 1 μL of mother liquor set against 1 mL reservoir solutions. 0.5 μL of seeding stock, prepared using Hampton seeding tubes (100 μL of 0.1 M Bicine/Tris buffer pH 8.5, 10% (w/v) PEG 4000, 25% (v/v) ethylene glycol) were added to each drop to promote crystallization. Typically, small crystals appeared overnight and were harvested on day four. G164E crystallized best in 0.1 M Bicine/Tris buffer (pH 8.5) containing 10% (w/v) PEG 4000, 25% (v/v) ethylene glycol and 30 mM D, L-lysine, and I379E in 55 mM D, L-lysine. The wild-type protein was crystallized with 10 mM NAD+ or 1 mM NADH (WT-NAD+ and WT-NADH) using the same buffer but without D, L-lysine. Crystals were flash-frozen and stored in liquid nitrogen. Diffraction data were collected at the Australian Synchrotron MX2 beam-line equipped with an ADSC Quantum 315r detector. G164E data were collected using a 20 μm micro-collimator with 0% beam attenuation, 5 s exposures and 1° oscillation angle, and I379E and WT-NAD+ data with 30% beam attenuation and 1 s exposures. Five WT-NADH datasets were collected from three crystals with varying attenuation (0–30%) and exposure times (1–2 s) and from a further small crystal using the micro-collimator. Data were processed using XDS. 130° of data from G164E were merged and scaled using Aimless in the CCP4 suite, and similarly 140° for I379E and 160° for WT-NAD+. For WT-NADH, 130°, 49°, 160°, 70° and 40° of each dataset were merged. Molecular replacement was performed using Phaser with a polyalanine WT model (PDB:4NWZ). The structures were refined using PHENIX with NCS restraints applied, COOT was used for model building and PyMOL to create the figures. Co-ordinates and structure factors have been deposited in the Protein Data Bank with Accession Numbers 5KMP, 5KMQ, 5KMR and 5KMS. […]

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