Computational protocol: CSL311, a novel, potent, therapeutic monoclonal antibody for the treatment of diseases mediated by the common β chain of the IL-3, GM-CSF and IL-5 receptors

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Protocol publication

[…] Data collection was carried out with a 20% attenuated beam at the MX2 beam line at the Australian Synchrotron. The crystals belonged to the C2 space group with unit cell dimensions of a = 99.95, b = 71.28, c = 221.23. Data were scaled and processed using XDS and Aimless and molecular replacement was carried out using Phaser (CCP4 suite). The βc ectodomain structure (PDB 2GYS) was modified to a partial βc dimer consisting of domains D4 and D1 only and was used as a search model to locate the βc molecule. The CSL311 Fab molecule was located using PDB 3HI5 as the search model (sequence similarity of ˜84% for heavy chain of CSL311) after deleting the CDR loops. A single solution comprising a partial βc dimer (comprising domains D1 and D2 from monomer A and domains D3 and D4 from monomer B) and one molecule of the CSL311 Fab comprising heavy (H) and light (L) chains was obtained with an Rwork/Rfree of 0.38/0.45 after first round of rigid body refinement. Initial electron density was clearest for the Fab molecule and domains D4 and D1 of βc, while density for βc domains D2 and D3 was the weakest. The initial βc/CSL311 model was further improved by iterative cycles of model building in Coot and model refinement using Phenix.Although the electron density maps for domains D4 and D1 of βc and the variable domains of Fab H and L chains was continuous, no electron density was observed for parts of D2 and D3 of βc and parts of the constant domains of the Fab H and L chains even at the end of refinement, suggesting that they might be either highly flexible or adopt multiple conformations. The structure was refined to a final Rwork/Rfree of 0.30/0.34. The structure was validated using Molprobity. The coordinates for the structure have been deposited with the RCSB Protein Data Bank (PDB 5DWU). […]

Pipeline specifications

Software tools XDS, CCP4, Coot, PHENIX, MolProbity
Applications Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma, Homo sapiens
Diseases Asthma
Chemicals Steroids