Computational protocol: Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

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Protocol publication

[…] Pm-SiaP was concentrated to 44 mg ml−1. Additional Neu5Ac was added to the protein at a 1:10 concentration ratio. Trays were set up at this concentration and crystals were grown by the hanging-drop vapour-diffusion method at 4°C using a Mosquito liquid-handling unit (a nanolitre dispensing robot; TTP Labtech). Drops were set up with equal volumes of protein and 1.6 M sodium citrate tribasic dihydrate pH 6.5 (crystallization buffer) and were suspended over 100 µl crystallization buffer. SiaP crystals were flash-cooled in mother liquor containing 5%(v/v) glycerol. The crystals were mounted in loops and X-ray diffraction data were collected on the IMCA-CAT 17-ID beamline at the Advanced Photon Source, Argonne, Ilinois, USA. The X-ray data were processed and scaled using XDS (Kabsch, 2010) and SCALA (Evans, 2011; Winn et al., 2011). [...] For all three structures (Pm-SiaP, Fn-SiaP and Vc-SiaP), molecular replacement was carried out using Phaser with the H. influenzae Neu5Ac-bound structure (PDB entry 3b50; Johnston et al., 2008) as the starting model. The starting model was generated from the PDB file (PDB entry 3b50) using CHAINSAW in the CCP4 package. This program retains the side chains of conserved residues but removes the atoms to the most conserved side-chain atom in the changed residues. All water molecules and ligands were removed. Refinement of the structures was performed using either PHENIX (Adams et al., 2011; for Pm-SiaP and Fn-SiaP) or REFMAC5 from the CCP4 package (for Vc-SiaP) and model building was carried out using Coot (Emsley et al., 2010). There is only one molecule in the asymmetric unit in all of the crystal forms. The two domains of the protein are tightly held together by the bound ligand in the structures of Fn-SiaP and Pm-SiaP. Hence, refinement of the domains by TLS did not reduce the R factor and R free in a statistically significant manner. However, we had expected that this would not be the case in the Vc-SiaP structure, as with no ligand bound there is a larger probability of observing domain motion-related effects. However, this was not the case and the primary reason for the lack of this could be the stabilization of the domains by intermolecular contacts mediated by the His tag and cobalt ion. Crystallographic data and refinement statistics are shown in Table 1. The structure and coordinates have been deposited in the PDB. The PDB code for Pm-SiaP is 4mmp, that for Fn-SiaP is 4mnp and that for Vc-SiaP is 4mag. […]

Pipeline specifications

Software tools XDS, CCP4, PHENIX, REFMAC5, Coot
Applications Small-angle scattering, Protein structure analysis
Organisms Fusobacterium nucleatum, Vibrio cholerae, Pasteurella multocida
Diseases Meningitis, Haemophilus, Pasteurella Infections
Chemicals Carbohydrates, Hydrogen, N-Acetylneuraminic Acid