Computational protocol: Structural and functional characterization of NanU, a novel high-affinity sialic acid-inducible binding protein of oral and gut-dwelling Bacteroidetes species

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Protocol publication

[…] Purified BF1720 was concentrated to 6 mg/ml in 25 mM sodium phosphate, pH 7.4, and tested for crystallization at 3 mg/ml with a variety of commercial screens. Subsequent optimization resulted in the growth of X-ray diffracting crystals grown with 1 μl of protein and 1 μl of reservoir solution [0.2 M ammonium acetate and 20% (w/v) PEG 3350] after 14 days of incubation at 17°C. Crystals were mounted direct from the drop without the addition of cryoprotectant and flash-cooled in a nitrogen gas stream maintained at 100 K. Data were collected to 1.6 Å on station I04-1 at the Diamond Light Source (Harwell, U.K.), and processed using XDS []. Molecular replacement was performed using the program PHASER on these data, employing a model obtained from the PHYRE2 server [] based upon a SusD superfamily protein from Bacteroides vulgatus (PDB code 3JQ0). The initial model was improved by application of the PIRATE and BUCCANEER programs from the CCP4 suite [] coupled to manual intervention in COOT [] interspersed with rounds of refinement using REFMAC []. A summary of the relevant data statistics is shown in Supplementary Table S3 (at Structural factors and co-ordinates have been deposited in the PDB under code 4L7T. All Figures were generated using PyMOL ( and the topology diagram was prepared using CorelDraw X4 (Corel Corporation).To ascertain whether both native and ligand-bound BF1720 were monomeric, 3 mg/ml of purified protein with and without pre-incubated Neu5Ac in 25 mM sodium phosphate, pH 7.4, at an equimolar concentration were sequentially applied to a HiLoad Superdex 200 PG gel-filtration column (GE Healthcare) at a flow rate of 1 ml/min. Apoferritin (443 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), BSA (66 kDa), ovalbumin (43 kDa), trypsin inhibitor (20 kDa), cytochrome c (12.3 kDa) and aprotinin (6.5 kDa) were used as molecular mass standards. […]

Pipeline specifications

Software tools XDS, Phyre, Buccaneer, CCP4, Coot, PyMOL, CorelDraw
Applications Miscellaneous, Small-angle scattering, Protein structure analysis
Organisms Escherichia coli, Bacteroides fragilis, Homo sapiens, Tannerella forsythia
Chemicals N-Acetylneuraminic Acid