Computational protocol: Crystallographic Study of Novel Transthyretin Ligands Exhibiting Negative-Cooperativity between Two Thyroxine Binding Sites

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Protocol publication

[…] A Mar345dtb image plate coupled to a Rigaku RU300 rotating anode X-ray generator, operating at 5 kW was used for data collection of all five complex structures and images were collected with an oscillation of 1° per image. A single crystal was placed in 33% glycerol containing mother liquor as a cryoprotectant and cooled to 120 Kelvin for diffraction experiments. Data reduction for all data sets were performed using MOSFLM and SCALA . Crystals of all TTR-ligand complex structures are isomorphous with the apo crystal form with the space group P21212 with two monomers in the asymmetric unit (). From the X-ray structure of apo wtTTR (PDB ID: 1DVQ ), chains A and B together, but without water molecules, ware used as an initial model for molecular replacement using the program PHASER . The initial protein atomic model was subjected to rigid body refinement using chains A and B as independent rigid body domains in REFMAC . The model was improved through alternated cycles of real space refinement using COOT , maximum likelihood minimization using restrained refinement protocols in REFMAC5 . Difference Fourier maps revealed distinct electron density for the ligand in each complex. The ligands were manually fitted in the unbiased omit maps which were calculated using the program CNS . Each of the ligand lies on the two fold symmetry on the crystallographic Z axis. For this reason, ligand molecules were initially refined with occupancy of 0.5. Water molecules were identified using find water module of COOT in the last steps of refinement. Online server MolProbity was used to check the quality of the refined structures. All the figures were prepared using PyMOL ( […]

Pipeline specifications

Software tools CCP4, Coot, REFMAC5, MolProbity, PyMOL
Application Protein structure analysis
Organisms Dipturus trachyderma
Diseases Amyloidosis
Chemicals Hydrogen, Thyroxine, Vitamin A