Computational protocol: Allosteric inhibition of the guanine nucleotide exchange factor DOCK5 by a small molecule

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Protocol publication

[…] All the data analyses were performed with programs from the ATSAS package unless specified otherwise and are summarized in Table . Radii of gyration (Rg) were evaluated by Guinier Wizard using the data within the range of Guinier approximation sRg < 1.3 and by Distance Distribution Wizard, both of which are modules of the PRIMUS program. The maximum distance Dmax was estimated with PRIMUS and refined by trial and error with GNOM. The distance distribution functions PI were calculated with GNOM. For DOCK5DHR2, we determined a range of Dmax values that accounted well for the experimental SAXS data. We then calculated DAMMIF models (see below) using Dmax values within this range, and selected the smallest Dmax value that accounted for the known 2-fold symmetry of DOCK5DHR2. The dimensionless Kratky plot was calculated by plotting (qRg)2I(q)/I(0) against qRg . The molecular weights of the proteins were estimated by the ScÅtter program (q = 0.25). The fit between scattering amplitudes calculated for crystal structures and the SAXS profiles were carried out with CRYSOl. ab initio shape determinations and structural envelopes calculations were done by DAMMIF, using the SAXS data within a q range of 0.01–0.2 Å−1. Initial calculations were carried out without imposing P2 symmetry and this resulted in models that were visually symmetrical. Given that all DockDHR2 proteins with known crystal structures are symmetrical dimers and DOCK5DHR2 is itself a dimer, subsequent models were constrained by a P2 symmetry to improve the quality of the models. 20 independent runs were carried out for each dataset. The resulting models were further compared and clustered by SUPCOMB. The Normalized Spatial Discrepancy (NSD) values were close to 1, which is in the high end for acceptable similarity between models but likely reflects the anisotropy of the structure. The model with the best fit to the experimental data was chosen for comparison with the crystal structure of Dock2DHR2. The PDB coordinates of the crystal structures were superimposed onto the SAXS-constructed envelopes of the proteins by SUPCOMB. SAXS data sets will be deposited with the small angle scattering biological data bank SASBDB (https://www.sasbdb.org/). [...] Crystallization screens were performed using the sitting-drop vapor diffusion method at 18 °C with a Mosquito robot (TTP LabTech) in 96-well crystallization plates by mixing 100 nL of Rac11–177-GDP at 5 mg/mL solution with 100 nL of precipitant solution. Crystals were obtained in 0.1 M MES pH 6.5 and 20% PEG 3350. Crystals were cryo-protected using the reservoir solution supplemented with 10% glycerol prior to flash freezing. A complete diffraction dataset was collected on PROXIMA-2A beamline (SOLEIL synchrotron, France) and was integrated with the program XDSme (https://code.google.com/archive/p/xdsme/). The structure was solved by molecular replacement with Phaser using Rac1-GPPNHP as a model. Refinement was carried out with the program BUSTER using TLS parameters, in alternation with graphical building using Coot. The quality of the structure was assessed using MolProbity. Data collection and refinement statistics are reported in Table . Coordinates and structure factors have been deposited in the Protein Data Bank with entry code 5N6O. […]

Pipeline specifications

Software tools ATSAS, DAMMIF, CRYSOL, Coot, MolProbity
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens, Mus musculus
Diseases Bone Diseases, Osteoporosis