Computational protocol: A modular interface of IL-4 allows for scalable affinity without affecting specificity for the IL-4 receptor

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Protocol publication

[…] Data for IL-4 or its variants were each obtained from a single crystal at 100 K at different beamlines (X06SA PX at the Swiss Light Source, Switzerland, ID14-1 at the European Synchrotron Radiation Facility, Grenoble, France) or a home source (Rigaku MicroMax007 with Osmic VariMax mirror system). The data were processed and integrated using the software MOSFLM version 6.2.1, and scaling was performed using SCALA CCP4 version 4.2.1; a summary of the processing statistics of the various datasets is presented in Table . To test for possible bias introduced by the model structure IL-4 (PDB entry 1HIK) used for data interpretation by the molecular replacement method, we also collected a dataset of a SeMet-labeled IL-4 variant F82D at three wavelengths (BW7A, EMBL DESY, Hamburg, Germany) (see Table ). Structures of the variant F82D refined by the MAD approach and molecular replacement were identical, showing that the model structure 1HIK used as the start structure for refinement did not bias the results of the individual variant structures. [...] The structures of IL-4 and the variants investigated in this study were refined using the lower resolution structure (PDB entry 1HIK) as a start model. To minimize possible bias through the start model structure, especially for the ill-defined loop regions, MAD phasing was applied to the IL-4 variant F82D. IL-4 (as well as the variant F82D) contains a single Met residue. The Seleno-Met site was determined and refined using the protocols supplied with the program CNS using a dataset measured at three wavelengths (inflection, peak and remote). The resulting electron density map was used to rebuild the loop regions between the first α-helix αA and the first short β-strand β1 (Glu19 to Cys24) as well as the long loops between β-strand β1 and helix αB (Lys37 to Glu41) and helix αc and the second β-strand β2 (Ser98 to Glu103). The resulting "improved" model was then used for interpretation of the diffraction data of the individual IL-4 proteins. The program REFMAC5 was used for subsequent refinement, followed by manual rebuilding of the models using the software QUANTA2000 (Accelrys Inc.). One TLS group was defined for the complete IL-4 molecule to account for anisotropy in the data. The progress of refinement was monitored by cross-validation using a test data set comprising 5% of the reflections. In the final refinement, Fobs – Fcalc difference electron density maps were used to identify water molecules as well as sulfate ions resulting from the high concentration of the ammonium sulfate precipitant. The final conventional and free R-factors for each model are presented in Table . […]

Pipeline specifications

Software tools iMosflm, CCP4, CNS, REFMAC5
Applications Small-angle scattering, Protein structure analysis
Diseases Asthma, Drug Hypersensitivity, Hypersensitivity