Computational protocol: Structural insights into the role of the Smoothened cysteine-rich domain in Hedgehog signalling

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Protocol publication

[…] An automated program UNIO was used for the NOE assignment and all these assignments were manually checked. Integrated NOE peaks were calibrated and converted to distance constraints with the program CALIBA. A total of 1421 meaningful distance constraints were derived from the NMR data and used as input for structure calculation using the program CYANA 2.1. The structure was refined using 139 dihedral angle constraints and 22 hydrogen bond constraints. The dihedral angles were generated by the program TALOS+ which uses six kinds (HN, HA, CA, CB, CO, N) of chemical shift values for a given residue as input to give the phi, psi angles. The Ramachandran statistics are as follows: most favourable 72.5 %, additionally allowed 25.7 %, generously allowed 1.6%, and disallowed 0.2 %. Also, four disulphide bonds were added as additional constraints between residues, C90–C155, C100–C148, C139–C179 and C172–C194. A total of 100 conformers were initially generated and 20 conformers with the lowest target function were used to represent the 3-D NMR structure. […]

Pipeline specifications

Software tools CYANA, TALOS+
Application NMR-based proteomics analysis
Organisms Drosophila melanogaster, Homo sapiens
Chemicals Budesonide