Computational protocol: Intrinsic Disorder of the C-Terminal Domain of Drosophila Methoprene-Tolerant Protein

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Protocol publication

[…] Disorder predictions were made using the IUPred server [,] available at Amino acid composition analyses were performed using the Composition Profiler available at [,]. The Uversky plot [], VSL2 [,], PONDR-FIT [], DISOPRED2 [] and FoldIndex [] calculations were made using PONDR at GeneSilico MetaDisorder server [] was used for results averaging. All analyses were performed using the default settings. [...] The concentration of purified protein was measured spectrophotometrically at 280 nm. The absorption coefficient of MetC (0.130 dm3·g−1·cm−1) was calculated using the ProtParam tool [], which is available at [...] SAXS experiments were performed on beamline P12 of the EMBL Hamburg Oustation at the Petra III storage ring at the Deutsches Elektronen-Synchrotron (DESY) in Hamburg []. Thirty-microlitre samples of MetC at a concentration of 2.6 mg/ml and buffer F (20 mM Tris-HCl and 150 mM NaCl) were loaded into a capillary cell using an automatic robotic sample changer []. All measurements were performed at 15°C. For each measurement, a total of 20 50-ms frames were recorded using a Pilatus 2M detector (Dectris, Switzerland). The Primus program [] from the ATSAS package [] was used for data processing, buffer subtraction and radius of gyration (Rg) value calculation. All SAXS data were collected over the scattering vector s range from 0.0025 to 0.441 Å-1. The pair distance distribution function p(r) was calculated using GNOM []. [...] The SAXS data were analysed using an ensemble optimization method (EOM) []. A pool of 10000 random conformers based on the MetC sequence was generated. A genetic algorithm was then applied to select an ensemble of models with the best fit to the experimental data []. The experimental SAXS data and MetC models from EOM have been deposited (SASBDB accession code: SASDBY5) at Small Angle Scattering Biological Data Bank ( []. […]

Pipeline specifications

Software tools IUPred, Composition Profiler, PONDR-FIT, DISOPRED, FoldIndex, Metadisorder, ProtParam
Databases ExPASy
Applications Protein structure analysis, Protein physicochemical analysis
Organisms Drosophila melanogaster
Diseases Dwarfism, Pituitary
Chemicals Ecdysterone, Methoprene