Computational protocol: Structural Analysis Uncovers Lipid-Binding Properties of Notch Ligands

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Protocol publication

[…] Crystals for the 2.5 Å apo structure were grown in 0.3 μl sitting drops using vapor diffusion at 3.2 mg/ml with 0.97 M sodium citrate (pH 5.0), 19.4% polyethylene glycol 6000 mother liquor at 25% after initial screening using commercially available reagents from Molecular Dimensions. Diffraction data were collected at the ESRF on beamline ID29 with a Pilatus 6M-F detector. The structure was solved by molecular replacement using the program Phaser and the previously published structure of Jagged1DSL-EGF123 (). The C2 domain was modeled using a combination of autobuild, buccaneer, and manual modeling ().Both calcium-bound forms were crystallized at 5.0 mg/ml with commercial reagents from Molecular Dimensions using the sitting-drop vapor-diffusion method with a drop size of 0.2 μl and 25% mother liquor. The 2.38 Å form was crystallized with 0.2 M sodium acetate, 0.1 M Bis-Tris propane (pH 7.5), and 20% polyethylene glycol 3350. The 2.84 Å form was crystallized using 0.2 M sodium iodide, 0.1 M Bis-Tris propane (pH 6.5), and 20% polyethylene glycol 3350. In addition, 10 mM CaCl2 was added to the initial protein solution, and the final concentration in the drop was 7.5 mM. Data were collected at the Diamond Synchrotron facility on beamline I03 with a Pilates 6M-F detector. Phases were determined by molecular replacement using the apo structure and the program Phaser. Refinement for all structures was performed using autobuster and COOT (). The density for the newly ordered loops is shown in prior to rebuilding. Data refinement and model statistics are summarized in . […]

Pipeline specifications

Software tools Buccaneer, Coot
Application Protein structure analysis
Organisms Drosophila melanogaster, Homo sapiens
Chemicals Calcium