Computational protocol: Crystal structure of PXY-TDIF complex reveals a conserved recognition mechanism among CLE peptide-receptor pairs

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Protocol publication

[…] Wild-type and various TDIF mutants were synthesized by Beijing Scilight Biotechnology LLC for crystallization and ITC assay. TDIF and PXYLRR were mixed at molar ratio of 5:1 for crystallization. Both the crystals of PXYLRR and PXYLRR-TDIF complex were obtained at 18 °C by the hanging-drop vapor-diffusion method. PXYLRR and PXYLRR-TDIF were crystallized in the same reservoir solution. The proteins were crystallized by mixing 1 μl each of the protein and a reservoir solution containing 2% v/v Tacsimate pH 5.0, 0.1 M Sodium citrate tribasic dihydrate pH 5.5, and 16% w/v Polyethylene glycol 3350. To prevent the crystals from radiation damage, all crystals were flash frozen using the reservoir buffer plus 15% glycerol as the cryoprotectant. All the diffraction data sets were collected at Shanghai Synchrotron Radiation Facility (SSRF) on the beam line BL17U1. All the data were processed using HKL2000 software package. The crystal structures of both PXYLRR and PXYLRR-TDIF complex were determined by molecular replacement performed with PHASER using the FLS2LRR structure (PDB code: 4MN8) as the initial search model. The models from MR were built with the program COOT and subsequently subjected to refinement by the program Phenix. Data collection, processing, and refinement statistics are summarized in . All the structure figures were prepared using PyMOL (DeLano, W. L. PyMOL Molecular Viewer. http://www.pymol.org, 2002). […]

Pipeline specifications

Software tools Coot, PHENIX, PyMOL
Application Protein structure analysis
Diseases Crohn Disease
Chemicals Amino Acids