Le X-ray scattering data collection and analysisSize-exclusion chromatography coupled tiny angle X-ray scattering (SEC-SAXS) data have been collected in the SAXS/WAXS beamline in the Australian Synchrotron [53] working with a sheath flow sample environment [54] at 12 keV (1.0332 A), making use of a detector distance of 1600 mm, and at a temperature of 293 K. Data were collected c-di-GMP (sodium);cyclic diguanylate (sodium);5GP-5GP (sodium) custom synthesis promptly immediately after elution from a Superdex S200 (5 150 mm) column at a flow price of 0.two ml.min-1 [55]. Samples were loaded on for the column at protein concentrations of eight.0, five.0 and 1.0 mg.ml-1 in buffer containing 50 mM bis-tris propane pH 7.5, 100 M cobalt chloride, 200 M PEP, five glycerol. Data were processed applying the reduction software ScatterBrain two.83, developed in the Australian Synchrotron. Scattering intensity (I) was plotted versus q, as a log-linear plot, and analysed using the ATSAS package [56]. Deconvolution on the information was accomplished using the HPLC module with the SOMO package [52,57] by fitting two pure Gaussian functions to each SEC-SAXS dataset. GASBOR [58] was made use of to generate ab initio dummy residue models from the P(r) obtained from the deconvoluted data for peaks A and B, which have been overlaid together with the crystal structure of PaeDAH7PSPA1901 (Protein Data Bank (PDB): 6BMC).Crystallography and structure determinationProtein crystals were prepared, by microbatch crystallisation [59], by mixing equal volumes of purified protein (final protein concentration three mg.ml-1 (6712 M)) with reservoir resolution (0.2 M sodium fluoride, 1 mM cobalt chloride, 1 mM PEP, 18 PEG 3350) and incubating at 278 K for 1 days. Crystals were flash frozen at 110 K in cryoprotectant containing 25 glycerol and mother liquor. X-ray diffraction information were collected at the Australian Synchrotron employing the MX2 beamline [60], equipped with an Eiger 16M detector, at a wavelength of 0.9536 A. DiffracPA1901 was solved by tion data have been processed applying XDS [61] and AIMLESS [62], plus the structure of PaeDAH7PS molecular replacement (MOLREP) [63] applying a single chain of PaeDAH7PSPA2843 (PDB: 5UXM) [33] because the search model. All ligands and waters have been removed in the search model before molecular replacement, as were residues corresponding for the inserted helices 2a and 2b . The sequence identity amongst the search model and the target protein was 43 . The model was constructed applying COOT [64] and refined with REFMAC [65].Interface analysisPISA [66] was applied to visualise and examine the residues involved in interface formation. LSQKAB [67] was utilized to superpose and examine the structures.PDB accession codesAtomic co-ordinates and structure aspects for the structure described in this work have already been deposited in the PDB with the accession code 6BMC.Benefits and discussionClustering of type II DAH7PS sequences reveals an uncharacterised subgroup of kind II enzymesClustering of form II DAH7PSs, determined by pairwise sequence similarity, enables the identification of two key clusters of sequences presenting higher intra- and low inter-cluster sequence Acetylcholinesterase ache Inhibitors Reagents similarity (Figure 2). The primary cluster consists of sequences corresponding to full-length type II DAH7PSs (which includes PaeDAH7PSPA2843 , MtuDAH7PS and CglDAH7PS) that contain each an N-terminal extension plus the 2a and 2b inserted helices. Nevertheless, a second distinct group of sequences, that are distant in the key cluster, is also evident. Sequences from this second grouping (of which PaeDAH7PSPA1901 is a member) are shorter in sequence length, relative to these found within the major.
