Computational protocol: Structural insights into Parkin substrate lysine targeting from minimal Miro substrates

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Protocol publication

[…] Crystals were grown using the sitting-drop vapor diffusion method at 21 °C with a drop size of 1 μL using a Phoenix robot (Art Robbins Instruments). Protein buffer solution contained: 25 mM HEPES, pH 7.4, 300 mM NaCl, 0.5 mM TCEP. For hMiro1-BC, Mg2+ and Ca2+ exchange was achieved by protein pre-incubation with protein buffer solution supplemented with 1 mM EGTA, followed by the addition of either 5 mM MgCl2 or CaCl2 and desalting into protein buffer solution supplemented with 5 mM Mg2+ or Ca2+. For hMiro1-BC nucleotide exchange, protein was pre-incubated with protein buffer solution supplemented with 1 mM EDTA, then incubated with 10 mM GDP or GMPPCP, applied to a desalting column and eluted into buffer containing 1 mM GDP or GMPPCP and 6 mM MgCl2. Crystallization conditions were as follows: hMiro1-BC_Ca2+ (10 mg/mL + 5 mM CaCl2 + 1 mM GDP): 0.04 M Potassium phosphate, 16% (w/v) PEG 8000, 20% (v/v) glycerol. hMiro1-BC_GDP (9 mg/mL + 5 mM MgCl2 + 1 mM GDP): 0.2 M Ammonium sulfate, 0.1 M tri-sodium citrate pH 5.6, 25% (w/v) PEG 4000. hMiro1-BC_GMPPCP (12 mg/mL + 6 mM MgCl2 + 10 mM GMPPCP): 0.2 M Ammonium sulfate, 0.1 M tri-sodium citrate pH 5.6, 17.5% (w/v) PEG 4000; hMiro1-C_C2221 (15 mg/mL + 6 mM MgCl2): 0.1 M Tris pH 8.5, 12% (w/v) PEG 4000. hMiro1-C_P41212 (12 mg/mL): 0.1 M Tris pH 8.5, 16% (w/v) PEG 10000. hMiro1-C_P3121 (19 mg/mL + 5 mM MgCl2): 0.1 M SPG buffer pH 5, 25% (w/v) PEG 1500. hMiro2-C (14 mg/mL): 0.2 M Magnesium Formate, 20% (w/v) PEG 3350. All crystals were harvested directly from their mother liquor without added cryoprotectants.Measurement of X-ray diffraction data was performed at the beamlines of the Life Sciences Collaborative Access Team (LS-CAT) at Sector 21 of the Advanced Photon Source in the Argonne National Laboratory. Data were measured at 100 K using a MarMosaic 225 CCD detector and were processed using the CCP4 suite. Molecular replacement was carried out in Phenix using fragments of the Drosophila Miro structure as search models (PDB ID 4C0L). Structures were interpreted and rebuilt using COOT. Crystallographic statistics for all structures are presented in . All figures were created in PyMol, Microsoft Excel, and/or Adobe Illustrator. The electrostatic surface potential representations () were generated using the PyMol APBS plugin. […]

Pipeline specifications

Software tools CCP4, PHENIX, Coot, PyMOL, Adobe Illustrator
Applications Miscellaneous, Small-angle scattering, Protein structure analysis
Organisms Homo sapiens