Computational protocol: Structures of the APC–ARM domain in complexes with discrete Amer1/WTX fragments reveal that it uses a consensus mode to recognize its binding partners

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Protocol publication

[…] Crystallization experiments were performed at 14 °C by the hanging-drop vapor-diffusion method. Crystals of the APC–ARM/Amer1-A1 complex were grown in 0.1 M MES, pH 6.5, and 12% PEG 20 000. Crystals of the APC–ARM/Amer1-A2 complex were obtained at 25% ethyleneglycol only. The APC–ARM/Amer1-A4 complex was crystallized under the condition of 0.1 M sodium/potassium phosphate, pH 6.2, 0.2 M NaCl, and 10% PEG 8000. Crystal diffraction data sets were all collected at the beamline BL17U1 at Shanghai Synchrotron Radiation Facility (China), and processed using the HKL2000 software [].Crystals of the APC–ARM/Amer1-A1 complex belonged to the P212121 space group, with one set of the complex in each asymmetric unit. The structure was determined at 1.90 Å by the method of molecular replacement with the CCP4 program Phaser [], using the structure of APC–ARM by itself (PDB code: 3NMW) [] as the searching model. After model-building by Coot [] and refinement by the CCP4 program REFMAC [, ], the final model has an R/R free factor of 18.06%/22.13%. In the Ramachandran plot, 99.4 and 0.6% of residues are in the most favored and allowed regions, respectively.Crystals of the APC–ARM/Amer1-A2 complex belonged to the P1 space group, with six sets of complexes in the asymmetry unit. The structure was determined at 2.10 Å using the same method as above. After refinement, the model has an R/R free factor of 19.34%/21.27%. In the Ramachandran plot, 99.1 and 0.9% of residues are in the most favored and allowed regions, respectively.Crystals of the APC–ARM/Amer1-A4 complex belonged to the P212121 space group, with one set of the complex in each asymmetry unit. The structure was determined at 1.70 Å using the same method as above. After refinement, the model has an R/R free factor of 19.67%/23.98%. In the Ramachandran plot, 99.1 and 0.9% of residues are in the most favored and allowed regions, respectively.The model qualities were all checked with the PROCHECK program []. [...] All protein structure figures were generated with PyMOL (http://www.pymol.org). […]

Pipeline specifications

Software tools CCP4, Coot, PROCHECK, PyMOL
Application Protein structure analysis