Computational protocol: Myelin 2′,3′-Cyclic Nucleotide 3′-Phosphodiesterase: Active-Site Ligand Binding and Molecular Conformation

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[…] For crystallization, the mouse CNPase catalytic domain was dialyzed into a buffer containing 10 mM sodium citrate pH 5.5, 50 mM NaCl, 10% glycerol, and 1 mM DTT. The detailed crystallization, soaking, and cryoprotection conditions are in . X-ray diffraction data were collected at synchrotron radiation beamlines BL14.1 (BESSY, Berlin) and I911-2 (MAX-Lab, Lund), at 100 K. Data were processed and scaled using XDS and XDSi . Molecular replacement was done using MOLREP or PHASER . The initial molecular replacement model was the catalytic domain of human CNPase . Later on, the best refined coordinates of mouse CNPase were always used. Structures were refined using PHENIX or REFMAC , and models were built using Coot . Simulated annealing omit maps in the absence of bound ligand were calculated in PHENIX. Processing and refinement statistics, as well as PDB accession numbers, are in .The Molprobity server was used for validation. Normal modes were calculated using the elastic network model . Structural homologs were detected using PDBeFold and Salami , and superpositions and structure analysis were carried out in Coot, ccp4mg , and PyMOL (http://www.pymol.org). [...] The molecular docking studies of ligands were performed with the crystal structure of CNPase. The product and water molecules in the PDB file were removed before the docking simulations. However, crucial water molecules forming hydrogen bonds with the product in the crystal structure were retained. The structures of docked ligands were obtained from the PDB and modified if necessary. The docking studies were performed with AutoDock Vina , with the aid of the AutoDock Tools interface. The side chains of several residues in the binding pocket of CNPase were set to be flexible. All rotatable bonds in the ligands were allowed to rotate freely. The grid map was centered on the binding pocket of the protein, and its size was set to 21×14×18 Å. The results were analyzed using PyMOL, and the final results were selected based on the binding modes . [...] All samples used for SAXS eluted from size exclusion chromatography as single symmetric peaks, corresponding to monomeric forms of the CNPase catalytic domain (see ). Initial synchrotron SAXS data were collected on the EMBL beamline ×33 at DESY, Hamburg, Germany. The CNPase catalytic domain was first transferred by size exclusion chromatography into a buffer containing 50 mM Bis-Tris-HCl pH 5.5, 100 mM NaCl, 3% glycerol, and 1 mM DTT. SAXS data were measured at 5–15 mg/ml. For analyzing the effects of citrate on the protein, SAXS data were collected on the ID14-3 beamline at ESRF, Grenoble. The buffer consisted of 20 mM Bis-Tris (pH 5.5), 300 mM NaCl, 1% glycerol, 1 mM TCEP, and concentrations between 1–10 mg/ml were used. Data were measured in the presence and absence of 50 mM sodium citrate. The comparison of the catalytic domain with N- and C-terminal extensions was carried out on the P12 EMBL BioSAXS beamline (PETRA III, DESY, Hamburg). The samples were in 20 mM Bis-Tris (pH 5.5), 200 mM NaCl, 1 mM TCEP. Sample concentrations ranged between 3 and 15 mg/ml.The data were analyzed with the ATSAS suite, as described , . PRIMUS was used for processing , GNOM for distance distribution evaluation , DAMMIN for ab initio model building , and DAMAVER for model averaging . […]

Pipeline specifications

Software tools AutoDock Vina, PyMOL
Application Protein interaction analysis
Organisms Mus musculus
Diseases Mitochondrial Diseases