On the need for an international effort to capture, share and use crystallization screening data

Development of an ontology for the description of crystallization experiments and results is proposed

A Novel Metagenomic Short-Chain Dehydrogenase/Reductase Attenuates Pseudomonas aeruginosa Biofilm Formation and Virulence on Caenorhabditis elegans

In Pseudomonas aeruginosa, the expression of a number of virulence factors, as well as biofilm formation, are controlled by quorum sensing (QS). N-Acylhomoserine lactones (AHLs) are an important class of signaling molecules involved in bacterial QS and in many pathogenic bacteria infection and host colonization are AHL-dependent. The AHL signaling molecules are subject to inactivation mainly by hydrolases (Enzyme Commission class number EC 3) (i.e. N-acyl-homoserine lactonases and N-acyl-homoserine-lactone acylases). Only little is known on quorum quenching mechanisms of oxidoreductases (EC 1). Here we report on the identification and structural characterization of the first NADP-dependent short-chain dehydrogenase/reductase (SDR) involved in inactivation of N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and derived from a metagenome library. The corresponding gene was isolated from a soil metagenome and designated bpiB09. Heterologous expression and crystallographic studies established BpiB09 as an NADP-dependent reductase. Although AHLs are probably not the native substrate of this metagenome-derived enzyme, its expression in P. aeruginosa PAO1 resulted in significantly reduced pyocyanin production, decreased motility, poor biofilm formation and absent paralysis of Caenorhabditis elegans. Furthermore, a genome-wide transcriptome study suggested that the level of lasI and rhlI transcription together with 36 well known QS regulated genes was significantly (≥10-fold) affected in P. aeruginosa strains expressing the bpiB09 gene in pBBR1MCS-5. Thus AHL oxidoreductases could be considered as potent tools for the development of quorum quenching strategies

A method for the general identification of protein crystals in crystallization experiments using a noncovalent fluorescent dye

A technique is described whereby the addition of low concentrations (millimolar to micromolar) of the fluorescent dye 1,8-ANS to the protein solution prior to crystallization results in crystallization experiments in which protein crystals are strongly contrasted above background artifacts when exposed to low-intensity UV radiation. As 1,8-ANS does not covalently modify the protein sample, no further handling or purification steps are necessary. The system has been tested on a wide variety of protein samples and it has been shown that the addition of 1,8-ANS has no discernible effect on the crystallization frequencies or crystallization conditions of these proteins. As 1,8-ANS interacts with a wide variety of proteins, this is proposed to be a general solution for the automated classification of protein crystallization images and the detection of protein crystals. The results also demonstrate the expected discrimination between salt and protein crystals, as well as allowing the straightforward identification of small crystals that grow in precipitate or under a protein skin

In situ protein crystal diffraction screening

The generation of high quality diffracting crystals remains an area that requires considerable person-power. Not only do crystals need to be produced and/or optimized, but such samples also have to be analyzed for their diffraction properties. While crystal shape and size are critical parameters controlling diffraction strength, diffraction screening remains the optimal manner to guide crystal growth optimization protocols. Mounting of the crystals to an appropriate sample support often requires transfer into a buffer significantly different from that the crystal is grown in, often resulting in a negative impact upon crystal morphology and diffraction properties. Modern synchrotron sources are increasingly using in situ exposure to X-rays to characterize protein crystals, with a growing number of structures being solved directly in situ, without recourse to manual handling of delicate samples. An automated approach requires crystal identification, positioning and collection of X-ray diffraction data for analysis. However, limitations are imposed by the variation in crystal size, morphology and space group. Here we review the availability and limitations of both commercial and synchrotron based infrastructures for in situ diffraction screening. We review the status of methods to establish the basic geometric features of crystals, the limitations currently inherent with in situ screening methods and we also describe our and other researchers efforts to overcome these limitations

In situ protein crystal diffraction screening

The generation of high quality diffracting crystals remains an area that requires considerable person-power. Not only do crystals need to be produced and/or optimized, but such samples also have to be analyzed for their diffraction properties. While crystal shape and size are critical parameters controlling diffraction strength, diffraction screening remains the optimal manner to guide crystal growth optimization protocols. Mounting of the crystals to an appropriate sample support often requires transfer into a buffer significantly different from that the crystal is grown in, often resulting in a negative impact upon crystal morphology and diffraction properties. Modern synchrotron sources are increasingly using in situ exposure to X-rays to characterize protein crystals, with a growing number of structures being solved directly in situ, without recourse to manual handling of delicate samples. An automated approach requires crystal identification, positioning and collection of X-ray diffraction data for analysis. However, limitations are imposed by the variation in crystal size, morphology and space group. Here we review the availability and limitations of both commercial and synchrotron based infrastructures for in situ diffraction screening. We review the status of methods to establish the basic geometric features of crystals, the limitations currently inherent with in situ screening methods and we also describe our and other researchers efforts to overcome these limitations