Computational protocol: Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors

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Protocol publication

[…] Crystals of muScn were obtained by hanging drop vapor diffusion at 25°C. The protein was concentrated to 12 mg/ml in standard phosphate-buffered saline (PBS) and mixed 1:1 v/v with a reservoir solution of 0.1 M HEPES (pH 7.5), 10% v/v isopropanol and 20% w/w PEG 4000. Crystals grew overnight and were cryoprotected in mother liquor containing 10% v/v glycerol. Diffraction data were collected at the Advanced Light Source 5.0.1 beamline (Lawrence Berkeley National Laboratory). Data were reduced with d*TREK (). Initial structure factor phase information was determined by molecular replacement, using the program PHASER () as a part of the CCP4i program suite (). The structure of human Siderocalin (PDB code: 1X89, chain A) was used as a search model. Model building and refinement were carried out using COOT () and refmac5 (). The structure was submitted to the TLSMD server (,) and TLS refinement using a single group was applied to the final model. Structure validation was carried out with the MolProbity server () and the RCSB ADIT server. […]

Pipeline specifications

Software tools Coot, REFMAC5, MolProbity
Application Protein structure analysis
Organisms Homo sapiens