Computational protocol: Crystal Structures of the Human G3BP1 NTF2-Like Domain Visualize FxFG Nup Repeat Specificity

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Protocol publication

[…] X-ray data was collected at beamline I911-2 at MAX-lab (Lund, Sweden). Data processing was carried out using the XDS pipeline in the xia2 software . PHASER , as implemented in the CCP4 suite , was used for initial structure determination of G3BP1 11–139 in space group P6322 by molecular replacement. The Cryptosporidium parvum nuclear transport factor 2 (PDB entry 1zo2, chain A ) was used as search model in the absence of a highly identical structure; PDB entry 3q90 became available significantly later. Experimental SIRAS phases obtained from a gold derivative using the program SHARP was used for improvement of electron density maps, but unambiguous tracing of the loops III and VII remained a challenge. Final refinement of the structure was performed using PHENIX software tools (phenix.autosol, phenix.den_refine and phenix.refine). Chain A of the G3BP1 NTF2-like domain in its FxFG peptide binding state (PDB: 4fcm) was used as a reference and for additional restraints in refinement. In addition, SIRAS phases from the gold derivative were used throughout as restraints for the MLHL target function. Automatic B-factor sharpening was applied in calculation of electron density maps before manual rebuilding in the program Coot . The phosphate included in the model occupies a special position and coordinates for its atoms were kept fixed in refinement and alternative orientations of the ion were ignored.The structures of G3BP1 1–139 with and without the FxFG peptide bound were solved in space group P212121 by molecular replacement and refined using the PHENIX software suite . An existing 1.7 Å crystal structure of the NTF2-like domain of human G3BP1 (PDB: 3q90) was used as search model. The program Coot was used for manual rebuilding and placement of the FxFG ligand, which was only included in the peptide complex after convergence of refinement. The structure models were validated using the program MolProbity and graphical figures were prepared in PyMOL (The PyMOL Molecular Graphics System, Version 1.3, Schrödinger, LLC). […]

Pipeline specifications

Software tools XDS, xia2, CCP4, PHENIX, Coot, MolProbity, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens
Diseases Neoplasms