Computational protocol: The Interaction of CCDC104/BARTL1 with Arl3 and Implications for Ciliary Function

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Protocol publication

[…] Native full-length Arl3 was purified and exchanged as previously described to be completely loaded with GppNHp (, ). Arl3∙GppNHp was mixed with BARTL1133 in a molar ratio of 1.3 to 1 at 16.7 mg/ml. The sitting-drop/vapor diffusion method was used, and initial conditions were established in EasyXtal CORE II Suite (1 M LiCl, 0.1 M MES [pH 6.0], 30% polyethylene glycol [PEG] 6000) and EasyXtal PEG II Suite (1 M LiCl, 0.1 M Tris [pH 8.5], 20% PEG 4000) from Qiagen. Crystals appeared after 1–3 days and were flash-frozen after 3 days from a 96-well screen in cryosolution containing the same constituents as the crystallizing condition supplemented with 20% glycerol. Crystals from the CORE II Suite were of space group P212121 and crystals from the PEG II Suite were of space group P21 (). Data were collected at the PXII X10SA beamline of the Swiss Light Source (SLS) and was indexed and processed with XDS (). Molecular replacement using different Arl structures was done with MOLREP and PHASER from the CCP4 package (). A model of the BARTL1133 sequence generated by the PHYRE threader based on BART (3DOE) was used in molecular replacement to solve the BARTL1133 structure in the complex. The structure was refined using REFMAC5 () to the following resolutions (Ramachandran statistics in parentheses): Arl3∙GppNHp∙BARTL1133 native P212121 to 2.2 Å (99.0% favored, 1.0% allowed, 0% outliers) and P21 to 2.0 Å (97.6% favored, 2.4% allowed, 0% outliers). Structures were deposited in the RCSB PDB databank with entry codes PDB: 4ZI2 and 4ZI3, respectively. For data and refinement statistics, see . All figures were produced using PYMOL (DeLano Scientific). […]

Pipeline specifications

Software tools Molrep, CCP4, Phyre, REFMAC5, PyMOL
Databases RCSB PDB
Application Protein structure analysis