Computational protocol: FUS-NLS/Transportin 1 Complex Structure Provides Insights into the Nuclear Targeting Mechanism of FUS and the Implications in ALS

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Protocol publication

[…] Hanging drops were made by mixing a solution (2 µl) containing the FUS-NLS/Trn1 complex (5 mg/ml protein in 20 mM HEPES, pH 7.3, 110 mM potassium acetate, 10 mM DTT) with an equal volume of reservoir solution containing 640 mM potassium-sodium tartrate and 20 mM HEPES buffer, pH 7.4. Crystals with a size of 200 µm × 50 µm × 10 µm were grown at 289°K within two weeks. Harvested crystals were cryoprotected with a reservoir solution supplemented with 26% (v/v) glycerol and then mounted for flash-cooling at 100°K. Diffraction data were collected at the beamline BL17U1 of Shanghai Synchrotron Radiation Facility (SSRF) (Shanghai, China) using an MX225 CCD detector. Data processing and reduction were carried out using the HKL2000 package . The structure of the FUS-NLS/Trn1 complex was solved first by molecular replacement with Molrep from CCP4 suite using the atomic coordinates of human Trn1 (PDB code: 2Z5J) as a search model. Molecular-replacement solutions were modified and refined with alternate cycles of manual refitting and building into a 2Fo − Fc composite omit electron density map using Coot and simulated annealing and maximum likelihood protocols using CNS , REFMAC , and phenix.refine . The final model of the complex was checked for geometrical correctness with PROCHECK . In the final model, the electron densities for residues 1–4 and 323–371 of the Trn1 and residues 495–507 of the FUS-NLS were invisible, and these 66 residues were excluded from the model. Furthermore, because of the poor electron densities for the side chains of residues K6, D8, R870 and R871 in Trn1, these 4 residues were mutated to Alanine in the model.Cartoon and surface representations were generated using PyMOL. The electrostatic potential was calculated and displayed with PyMOL. The atomic coordinates and structural factors for the human Trn1/FUS-NLS complex have been deposited in the Protein Data Bank (PDB) database with accession code 4FQ3. […]

Pipeline specifications

Software tools Molrep, CCP4, Coot, CNS, PHENIX, PROCHECK, PyMOL
Application Protein structure analysis
Organisms Homo sapiens
Diseases Amyotrophic Lateral Sclerosis, Sarcoma