Computational protocol: Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding*

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Protocol publication

[…] Protein fluorescence experiments were performed in a 3-ml quartz cuvette (Starna) using a FluoroMax2 (Instruments SA, Inc.) with an LTD6 water bath (Grant) controlled with the supplied software, DataMax-Std version 2.20. SiaP contains no tryptophans, and so the protein was excited for tyrosine fluorescence. 0.05 μm protein in 50 mm Tris/HCl, pH 8.0, was excited at 281 nm with slit widths of 5–10 nm to give a signal intensity of 2–3 × 106 units. Ligand was added at concentrations and times to produce spectra and time course titrations. For titrations, the cumulative fluorescence change was plotted using SigmaPlot (version 10.0), and the Kd value was determined using a fit to a simple hyperbolic curve. For the F170W/R147K mutant titrations, protein was used at 0.5 μm. To obtain the required millimolar additions of Neu5Ac for titrations, additions of 40 μl of ligand were added, and so a dilution only control was run in parallel to remove dilution effects from the calculation of KD values. [...] SiaP WT and mutant proteins at a concentration of 30–35 mg/ml were co-crystallized with the respective ligand at concentrations of 10, 15, and 20 mm (for R147K, R147A, and R147E/WT, respectively) using 300-nl sitting drops in 96-well Greiner plates composed of an equivolume of protein and reservoir buffer (100 mm MES, pH 6.0, 28.5% PEG 6K (w/v), with varying additives). Crystals obtained at 4 °C were vitrified in the presence of 10% glycerol and tested in-house before data collection at ID23-2 at the ESRF synchrotron facility in Grenoble, France, and at beamline I04 at the DIAMOND Lightsource, Didcot, UK.Data were processed in MOSFLM and SCALA, and initial phases were obtained by rigid body refinement using 2V4C as a closed model and for the 5% Rfree batch. The WT-Neu5Ac structure was solved using ACORN ab initio phasing to eliminate model bias at atomic resolution with a fragment subset of 800 atoms starting from atom 1 of the same model. Cycles of refinement within Refmac (version 5.5.0109) were iterated with model building in Coot (version 12). Ligand and water molecules were added in the final iterations of model building and refinement, and Babinet scaling was applied. Structures were refined anisotropically with the exception of the R147K structure (2xwi), which was refined isotropically at a resolution of 2.2 Å. Composite omit maps were calculated with Comit () for the placement of low occupancy water molecules that were added automatically using Coot. Finally, water molecules were checked to fulfill the following criteria: the “Density Fit Graph” and “Check Waters” validation tools in Coot, B-factor variance, and H-bond distances. The identity of the compounds within the WT and R147E structure was checked using unrestrained refinement on the basis of their atomic B-factors and bond lengths. Finally, the data were checked using Coot, Sfcheck, Procheck, and the ADIT Protein Data Bank deposition server. The superpositions and figures were generated with PyMOL version 0.99 and CorelDRAW X3, respectively. […]

Pipeline specifications

Software tools SigmaPlot, CCP4, PROCHECK, PyMOL, CorelDraw
Applications Miscellaneous, Protein structure analysis
Organisms Haemophilus influenzae
Chemicals N-Acetylneuraminic Acid