Computational protocol: Insights into the inhibitory mechanisms of NADH on the αγ heterodimer of human NAD-dependent isocitrate dehydrogenase

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Protocol publication

[…] To obtain the αγ heterodimer bound with NADH, the protein solution (10 mg/ml) was incubated with NADH (final concentration of 10 mM) at 4 °C before crystallization. Crystallization screening was carried out using the hanging drop vapor diffusion method, and crystals of the αMg+NADHγNADH heterodimer were grown at 20 °C from drops consisting of 1 μl each of the protein solution and the reservoir solution [0.1 M HEPES (pH 7.5), 50 mM MgCl2, and 30% (v/v) PEGMME 550]. Diffraction data were collected from a flashed-cooled crystal at 100 K at beamline 19U1 of NFPSS, China, and processed, integrated and scaled together using HKL3000. The flashed-cooled crystal was prepared by soaking the crystal into the cryo-protectant containing the reservoir solution mixed with 20% ethylene glycol and then quickly dipping into liquid N2. Crystallographic statistics are listed in Table .The αMg+NADHγNADH structure was solved with the molecular replacement (MR) method as implemented in program Phaser using the αMgγ structure (PDB code 5GRH) as the search model. Structure refinement was performed using programs Phenix and REFMAC5, and manual model building with program Coot. Structural analyses were performed using programs in the CCP4 suite and the PISA server. The structure figures were generated using PyMol and the structure-based sequence alignment figure using ESPpript 3.0. Statistics of the structure refinement and the quality of the structure model are also listed in Table . […]

Pipeline specifications

Software tools PHENIX, REFMAC5, Coot, CCP4, PyMOL
Application Protein structure analysis
Organisms Homo sapiens
Chemicals Adenosine Diphosphate, NAD, Citric Acid