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 (www.rcsb.org) with accession number 4U9T. […]

Pipeline specifications

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