Computational protocol: Structural and functional analysis of the GABARAP interaction motif (GIM)

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[…] The PLEKHM1629–638‐LC3A2–121, PLEKHM1629–638‐GABARAP2–117 and PLEKHM1629–638‐GABARAP‐L12–117 chimeric proteins were purified and crystallized as N‐terminally LIR‐fused chimeric proteins. The LC3C8–125 protein was co‐crystallized with the PLEKHM1‐LIR peptide (GAMG‐P629QQEDEWVNVQYPD642). Initial crystallization trial was performed using Hampton Research (Crystal screen, Crystal screen cryo, Index and PEG/Ion) and Molecular dimension (JCSG+, Midas, Morpheus, PACT, Clear Screen Strategy 1 and Clear Screen Strategy 1). In all cases, the drops included 400 nl of protein (concentrations listed below) and 400 nl of mother liquor. All crystallization experiments were set up at 4°C.For PLEKHM1629–638‐LC3A2–121 (10 mg ml−1), crystals were grown in the JCSGplus screen condition H7 (0.2 M ammonium acetate, 0.1 M Bis Tris, pH 5.5, 25% w/v polyethylene glycol 3,350). Crystals for PLEKHM1629–638‐GABARAP2–117 (9.1 mg ml−1) were grown in the PEG/ion screen condition F5 (4% v/v Tacsimate pH 8.0, 12% w/v polyethylene glycol 3,350). Crystals for the PLEKHM1629–638‐GABARAP‐L12–117 protein (7.5 mg ml−1) were formed in the PEG/ion screen condition A6 (20% w/v polyethylene glycol 3,350, 0.2 M NaCl, 8% MPD pH 7.2). The LC3C8–125 protein (9.2 mg ml−1) was mixed with the PLEKHM1 peptide (2.4 mg ml−1) in equal volume and incubated for 3 h at 4°C, prior to setting up the crystallization trays. Crystals were formed in the PEG/ion screen condition D5 (0.2 M potassium phosphate monobasic, 20% w/v polyethylene glycol 3,350). The crystals were frozen in liquid N2 prior to data collection.X‐ray diffraction data were collected on the MX2 microcrystallography beamline at the Australian synchrotron (Melbourne, Australia). The data were integrated using XDS and scaled using Aimless . The PLEKHM1629–638‐GABARAP2–117 and PLEKHM1629–638‐GABARAP‐L12–117 structures were solved by molecular replacement using MOLREP and search models 1GNU and 2R2Q, respectively. Phases for the PLEKHM1‐LIR:LC3C co‐crystal structure were estimated using PHASER and the search model was 3WAM. The solved structures were refined using PHENIX.REFINE , and manual refinement was performed using COOT . The images in the work were generated using PyMOL (The PyMOL Molecular Graphics System, Version Schrödinger, LLC). […]

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