Computational protocol: Structural basis of JAZ repression of MYC transcription factors in jasmonate signaling

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Protocol publication

[…] Data collections were performed at sector 21-ID (LS-CAT) beam lines of the APS synchrotron using single native MYC3(5-242) crystals, MYC3(44-238)-Jas22JAZ9 complex crystals, and Se-Met substituted MYC3(44-238) crystals at different wavelengths. All diffraction data were processed using XDS, and merged using Aimless of the CCP4 suite. Initial phasing was tried by using the single wavelength anomalous dispersion (SAD) method based on anomalous diffraction of sulfur atoms as previously described. S-SAD phasing using a combined data set of 11 native MYC3(5-242) crystals collected at 1.77 Å was not successful, probably due to the non-isomorphism of the individual crystals. To solve the phase problem, the Se-Met substituted MYC3(44-238) crystals were prepared and a single data set was collected at a peak wavelength of 0.9787 Å (). Se-SAD phasing was performed by using the Phenix Autosol program. Five out of six selenium atoms were found with an FOM of 0.41. The Phenix autobuild program generated an initial model of 286 residues with an Rwork/Rfree of 0.35/0.40. Further model improvements were performed using Coot and refined using the Refmac program in CCP4 to a final model with an R factor of 0.21 and an Rfree factor of 0.26. The MYC3(5-242) apo structure, MYC3(5-242)–Jas22JAZ9, MYC3(44-238)–Jas22JAZ9 and MYC3(44-238)–Jas22JAZ1 complex structures were solved by using the molecular replacement program Phaser with the Se-Met MYC3(44-238) structure as a search model. The model for the α1’/ α1 helices for the MYC3(5-242) structure, for the Jas22JAZ9 peptide for the MYC3(44-238)–Jas22JAZ9 and MYC3(5-242)–Jas22JAZ9 complex structures and for the Jas22JAZ1 peptide for the MYC3(44-238)–Jas22JAZ1 complex structure were built based on the electron density maps using Coot. […]

Pipeline specifications

Software tools XDS, CCP4, PHENIX, Coot
Applications Small-angle scattering, Protein structure analysis
Organisms Arabidopsis thaliana