Computational protocol: Crystal structures of the human Dysferlin inner DysF domain

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Protocol publication

[…] The inner DysF domain was screened for crystallisation at 16°C, by the sitting-drop method in 96- well crystallisation plates (Molecular Dimensions), using PACT screen []. After 11 days, small crystals appeared in a drop with mother liquor consisting of 0.2 M NaBr and 20% PEG 3350. This crystallisation could not be reproduced initially. 0.2 M NaBr was then added to the protein sample and the crystallisation screen was done again. Bigger crystals were produced in one day, where the reservoir contained 0.04 M potassium dihydrogen phosphate, 16% w/v PEG 8000 and 20% v/v Glycerol. 0.3 mm cubic crystals were grown by the hanging drop method based on this condition. The crystal was cryoprotected in crystallisation buffer with the glycerol concentration increased to 25%. Initial diffraction data was collected to 2.3 Å in house using MicroMax TM -007 rotating anode X-ray generator (λ = 1.54 Å) and Saturn 944+ CCD detector with Varimax optics. Further data was collected at Soleil beamline proxima 1 and at beam ID29 at ESRF. Diffraction images were processed using XDS software package [], and scaled using aimless in the CCP4 program suite []. The initial phases of the dysferlin inner DysF domain were determined by molecular replacement with the program Mr Bump [] using human myoferlin inner DysF domain [] [PDB:2K2O] as the search model. The model was manually rebuilt in COOT [] and refined initially in Refmac [] and then continued in PHENIX []. Data collection and refinement statistics are summarized in Table .No human subjects were directly used in this study. Human mutation data was taken from publicly accessible databases. […]

Pipeline specifications

Software tools XDS, Coot, PHENIX
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens
Diseases Muscular Dystrophies