Computational protocol: Structural and functional analysis of MiD51, a dynamin receptor required for mitochondrial fission

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Protocol publication

[…] Crystals of MiD51 and MiD51 complexes were obtained by hanging drop vapor diffusion in 16% (wt/vol) polyethylene glycol 8000, 0.10 M Hepes, pH 7.5, 10.0% (vol/vol) 2-propanol, and 0.2 M ammonium sulfate at 293 K by combining 1 µl of protein with 1 µl of crystallant over 500 µl of reservoir solution. Co-crystallization of nucleotides was performed by the addition of 10 mM nucleotide and 20 mM MnCl2, followed by flash-cooling in mother liquor supplemented with 15% glycerol, 0.02 M nucleotide, and 20 mM MnCl2. Xenon derivatization of native crystals was performed using a xenon chamber (Hampton) at the Australian Synchrotron. Native and derivative data were collected at the Australian Synchrotron, Beamline MX2, at 100 K and wavelengths of 0.9537 and 1.4586 Å, respectively. Initial experimental phases of native MiD51ΔN118 were obtained using Xe-SAD in PHENIX Autosol. Secondary structure elements were identified from the initial experimental map and modeled using polyalanine chains. Initial polyalanine models were used to improve xenon sites until the entire MiD51 chain could be traced. The polyalanine model was subsequently improved using automated building in Buccaneer (, ), followed by manual rebuilding using Coot. The final model was refined to 2.0 Å with a final Rwork and Rfree of 0.218 and 0.266, respectively.MiD51–GDP and MiD51–ADP complexes were solved by molecular replacement using nonnucleotide bound MiD51 as a search model. Crystallization of MiD51ΔN118/PEYFP in complex with GDP and ADP was achieved in 1.0 M sodium malonate, pH 6.0, 0.01 M nucleotide, and 0.02 M MnCl2. Crystals were cryoprotected in 15% glycerol and data were collected at 1.4586 Å for anomalous signal determination. The structure was solved by molecular replacement using a native MiD51 monomer as a search model. Refinement of all structures was performed using PHENIX (). MolProbity validation revealed no residues in disallowed Ramachandran plot regions. Final crystallographic data and refinement statistics are summarized in . […]

Pipeline specifications

Software tools PHENIX, Buccaneer, Coot, MolProbity
Application Protein structure analysis
Organisms Homo sapiens
Diseases Nervous System Diseases
Chemicals Adenosine Diphosphate, Guanosine, Guanosine Diphosphate, Nucleotides