Computational protocol: Flexible Structure of Peptide-Bound Filamin A Mechanosensor Domain Pair 20–21

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Protocol publication

[…] SAXS data were collected at the European Synchrotron Radiation Facility (Grenoble, France), beamline BM29 [] (). The data were collected at 277 K in 20 mM Tris (pH 8.0), 100 mM NaCl, and 10 mM DTT using 1–4 mg/ml concentrations of IgFLNa20–21 and IgFLNa∆A20–21. Two times molar excess of the migfilin peptide was used to ensure saturation in binding. The small size of the peptide enabled direct subtraction of its scattering when applied also to the sample buffer. A PILATUS 1M image plate was used, at a sample/detector distance of 2.85 m and wavelength of 0.10 Å, covering the momentum transfer range of 0.01 < q < 5 nm-1 (q = 4ᴨsin(θ)/λ where 2θ is the scattering angle). The data were processed using the standard procedures of the ATSAS program package []. Buffer subtractions were conducted with PRIMUS []. The radius of gyration R g was estimated with AUTORG [] and distance distribution functions P(r) and particle maximum dimension D max were estimated using DATGNOM []. DATPOROD was used to estimate the excluded volume (V p) of the hydrated particle []. Dimensionless Kratky (qR g 2 x I(q)/I(0) versus qR g for V c normalized and qV c 2 x I(q)/I(0) versus qV c for volume-of-correlation V c normalized) [] and Porod–Debye (q4 x I(q) versus q4) [] plots were used to assess the flexibility of the proteins. V c was calculated with the program SCÅTTER []. The bead-modeling program DAMMIF [] was used to generate an ab initio model of IgFLNa20–21. Ten individual runs of DAMMIF were performed and averaged with DAMAVER []. CRYSOL [] was used to evaluate the scattering of the IgFLNa20–21 crystal structure (from IgFLNa19–21 crystal structure, PDB ID: 2J3S) []. SUPCOMB [] was used to overlay the crystal structure and the ab initio model with minimal normalized spatial discrepancy. An ensemble optimization method (EOM) [] was used to further model the inter-domain flexibility and size distribution in solution. First, a pool of 10,000 randomly generated models of IgFLNa20–21 and IgFLNa∆A20–21 were generated with the RanCh program. The inter-domain linker (residues 2230–2236) and IgFLNa20 A strand (residues 2141–2150) were considered to be random chains. Then, a genetic algorithm program, GAJOE, was used to select an ensemble of 20 models whose combined scattering best fit with the experimental scattering. The data along with the ab initio model were submitted to SASBDB []. [...] IgFLNa∆A20–21 in complex with migfilin peptide (5PEKRVASSVFITLAPPRR DVAVAE28, EZBiolab, Westfield, IN) was crystallized using the hanging drop vapor diffusion method at 295 K with an equimolar (1 mM) protein-peptide mixture. Next, 2 μl droplets containing equal volumes of the protein-peptide mixture and 0.1 M MES at pH 6, 1.9 M (NH4)2SO4, 0.1 M (CH3CO2)3Pr were equilibrated against 1 ml of the reservoir solution. The crystals were transferred to 25% glycerol in the reservoir solution before freezing under liquid nitrogen. The data were collected at 100 K at the European Synchrotron Radiation Facility (Grenoble, France), beamline ID14–1, using the ADSC Q210 CCD detector, and were processed using the XDS program package []. The crystal structure was solved by molecular replacement with Phaser [] using the structure of IgFLNa21 (PDB code: 2W0P, A chain) [] as a search model. The model was built using ARP/wARP [] and Coot [] and refined using REFMAC 5.5 []. TLS refinement parameters were defined with help of the TLSMD server []. Final refinement was made using the PDB-REDO server to optimize the refinement parameters []. Structural factors and atomic coordinates were deposited in the PDB with ID 4P3W. All crystallographic figures were generated with PyMOL (Schrödinger LCC, Portland, OR). […]

Pipeline specifications

Software tools ATSAS, DAMMIF, CRYSOL, EOM, XDS, ARP/wARP, Coot, PyMOL
Databases PDB_REDO
Applications Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma