Computational protocol: Distinct N-terminal regions of the exomer secretory vesicle cargo Chs3 regulate its trafficking itinerary

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Protocol publication

[…] The Chs5(1–77)/Chs6-6xHis exomer complex was co-crystallized with Chs3 peptides (Genscript, 95% purity) using the hanging drop vapor diffusion method. The peptides were resuspended in the precipitant solution (0.3 M ammonium sulfate, 0.1 M citric acid pH 4.0) to a concentration of 100 μ M. 1 μl of the peptide solution was mixed with 1 μl of 25 mg/ml exomer, resulting in a molar ratio of 3.85:1 peptide:exomer, and this drop was placed on a cover slip above the precipitant solution. Hexagonal plate-shaped crystals appeared after 5–7 days. Crystals were cryoprotected in 0.3 M ammonium sulfate, 0.1 M citric acid pH 4.0, 30% glycerol, and 100 μ M peptide. Diffraction data were collected at CHESS (Cornell High Energy Synchrotron Source) beamline A1 and processed using HKL-2000 (Otwinowski and Minor, ). The structure was solved by molecular replacement with Phaser in the PHENIX software suite (Adams et al., ) using residues 1–77 of Chs5 and all of Chs6 from the Chs5(1–299)/Chs6 exomer complex structure (PDB: 4GNS; Paczkowski et al., ). Density for the Chs3 peptide was clearly visible in initial difference maps (Figure 6A). The model was refined by manual adjustment in Coot (Emsley et al., ) and refinement in PHENIX. Our software is maintained by SBGrid (Morin et al., ).The coordinates and structure factors have been deposited in the Protein Data Bank ( with accession number 4U9T. […]

Pipeline specifications

Software tools HKL-2000, PHENIX, Coot
Application Protein structure analysis