Computational protocol: Structural Basis for the Interaction of Unstructured Neuron Specific Substrates Neuromodulin and Neurogranin with Calmodulin

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Protocol publication

[…] Crystallization trails for apo CaM-(Gly)5-NmIQ and apo CaM-(Gly)5-NgIQ complexes were performed with a protein concentration of 10–12 mg/ml using the hanging drop vapor diffusion method at room temperature. The initially identified conditions from Hampton Research (Aliso Viejo, CA, USA) and Qiagen (Valencia, CA, USA) were further optimized. Best crystals of apo CaM-(Gly)5-NmIQ complex crystals were obtained from a condition consisting of 0.12 mM MgAcO, 8% PEG 3350 and 10% EtOH. Similarly, the apo CaM-(Gly)5-NgIQ complex crystals were obtained using a condition consisting of 0.1 M imidazole (pH 8.0) and 1.2 M sodium citrate tribasic dihydrate. Where necessary, crystals were cryo-protected with reservoir condition supplemented with 10% glycerol and flash-cooled in N2 cold stream at 100 K.The molecular replacement method did not yield any structure solution, which led us to collect the Single wavelength Anomalous Dispersion (SAD) data sets in the synchrotron beam line X8C (NSLS, Brookhaven National Laboratory) and 13B1 SW6 (National Synchrotron Radiation Research Center (NSRRC), Taiwan) using a Quantum 4-CCD detector (Area Detector Systems Corp Poway, CA, USA). All data sets were processed using HKL2000. Heavy atom (Se) location, phasing and density modification were performed using the program ShelxC/D/E, and model building was carried out with the program Buccaneer in CCP4. Where necessary, the model was manually built in COOT and twin refinement was carried out in Refmac5. At the final stage of refinement, well-ordered water molecules were included. The models have good stereochemistry, as analyzed by PROCHECK (). All structure-related figures reported in this manuscript were generated using PyMol. […]

Pipeline specifications

Software tools SHELX, Buccaneer, Coot, REFMAC5, PROCHECK, PyMOL
Application Protein structure analysis
Diseases Protein C Deficiency