Computational protocol: Structural and Functional Characterization of Cargo-Binding Sites on the μ4-Subunit of Adaptor Protein Complex 4

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Protocol publication

[…] Unless otherwise stated, solutions and crystallization reagents were from Hampton Research (Aliso Viejo, CA). Crystals of the C-terminal domain of μ4-D190A in complex with the APP peptide ENPTYKFFEQ (New England Peptide) were grown by the hanging drop method at 21°C. Prior to crystallization, the protein was incubated at room temperature for 1 h with 2.5 mM peptide. The reservoir solution contained 0.1 M HEPES (pH 7.0) and 15% (w/v) PEG 6000. Hanging drops were set up by mixing 1 µl of reservoir solution with 2 µl of preincubated protein-peptide complex (10 mg/ml). Under these conditions crystals appeared after 24 h. Crystals were cryoprotected in the reservoir solution supplemented with 20% glycerol and then flash-frozen in liquid nitrogen. The complex crystallized in space group P21 and crystals diffracted up to 1.84 Å. A native data set was collected from a single crystal at the SER-CAT beamline 22-ID-D, equipped with a MAR CCD detector (Advanced Photon Source, Argonne National Laboratory). Data were processed using HKL2000 . Data collection statistics are shown in . The structure was determined by molecular replacement with wild-type μ4 without ligands as search model (pdb entry 3L81; ) using the program Phaser , as implemented in the ccp4 suite of programs for protein crystallography . Iterative manual model building and initial refinement were done using COOT and REFMAC . The final model comprises 269 residues of μ4, the residues TYKFFEQ of APP, and 97 water molecules. Figures were prepared in MacPyMol (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC). Crystallographic coordinates and structure factors have been deposited with the Protein Data Bank (accession code 4MDR). […]

Pipeline specifications

Software tools CCP4, Coot, PyMOL
Application Protein structure analysis
Diseases Alzheimer Disease