The structure of SAV1646 from Staphylococcus aureus belonging to a new `ribosome-associated' subfamily of bacterial proteins.

The crystal structure of the SAV1646 protein from the pathogenic microorganism Staphylococcus aureus has been determined at 1.7 Šresolution. The 106-amino-acid protein forms a two-layer sandwich with α/ß topology. The protein molecules associate as dimers in the crystal and in solution, with the monomers related by a pseudo-twofold rotation axis. A sequence-homology search identified the protein as a member of a new subfamily of yet uncharacterized bacterial `ribosome-associated' proteins with at least 13 members to date. A detailed analysis of the crystal protein structure along with the genomic structure of the operon containing the sav1646 gene allowed a tentative functional model of this protein to be proposed. The SAV1646 dimer is assumed to form a complex with ribosomal proteins L21 and L27 which could help to complete the assembly of the large subunit of the ribosome.

X-CHIP: an integrated platform for high-throughput protein crystallization and on-the-chip X-ray diffraction data collection.

The X-CHIP (X-ray Crystallization High-throughput Integrated Platform) is a novel microchip that has been developed to combine multiple steps of the crystallographic pipeline from crystallization to diffraction data collection on a single device to streamline the entire process. The system has been designed for crystallization condition screening, visual crystal inspection, initial X-ray screening and data collection in a high-throughput fashion. X-ray diffraction data acquisition can be performed directly on-the-chip at room temperature using an in situ approach. The capabilities of the chip eliminate the necessity for manual crystal handling and cryoprotection of crystal samples, while allowing data collection from multiple crystals in the same drop. This technology would be especially beneficial for projects with large volumes of data, such as protein-complex studies and fragment-based screening. The platform employs hydrophilic and hydrophobic concentric ring surfaces on a miniature plate transparent to visible light and X-rays to create a well defined and stable microbatch crystallization environment. The results of crystallization and data-collection experiments demonstrate that high-quality well diffracting crystals can be grown and high-resolution diffraction data sets can be collected using this technology. Furthermore, the quality of a single-wavelength anomalous dispersion data set collected with the X-CHIP at room temperature was sufficient to generate interpretable electron-density maps. This technology is highly resource-efficient owing to the use of nanolitre-scale drop volumes. It does not require any modification for most in-house and synchrotron beamline systems and offers a promising opportunity for full automation of the X-ray structure-determination process.

Crystal structure of alkyl hydroperoxidase D like protein PA0269 from Pseudomonas aeruginosa: homology of the AhpD-like structural family.

BACKGROUND: Alkyl hydroperoxidase activity provides an important antioxidant defense for bacterial cells. The catalytic mechanism requires two peroxidases, AhpC and AhpD, where AhpD plays the role of an essential adaptor protein. RESULTS: The crystal structure of a putative AhpD from Pseudomonas aeruginosa has been determined at 1.9 Å. The protein has an all-helical fold with a chain topology similar to a known AhpD from Mycobacterium tuberculosis despite a low overall sequence identity of 9%. A conserved two α-helical motif responsible for function is present in both. However, in the P. aeruginosa protein, helices H3, H4 of this motif are located at the N-terminal part of the chain, while in M. tuberculosis AhpD, the corresponding helices H8, H9 are situated at the C-terminus. Residues 24-62 of the putative catalytic region of P. aeruginosa have a higher sequence identity of 33% where the functional activity is supplied by a proton relay system of five residues, Glu36, Cys48, Tyr50, Cys51, and His55, and one structural water molecule. A comparison of five other related hypothetical proteins from various species, assigned to the alkyl hydroperoxidase D-like protein family, shows they contain the same conserved structural motif and catalytic sequence Cys-X-X-Cys. We have shown that AhpD from P. aeruginosa exhibits a weak ability to reduce H(2)O(2) as tested using a ferrous oxidation-xylenol orange (FOX) assay, and this activity is blocked by thiol alkylating reagents. CONCLUSION: Thus, this hypothetical protein was assigned to the AhpD-like protein family with peroxidase-related activity. The functional relationship of specific oligomeric structures of AhpD-like structural family is discussed.