Publications

Plastic microchannel crystallization template designs made from inexpensive cyclic olefin copolymers have been shown to be low-birefringent, X-ray transmissive and compatible with microfluidic fabrication in restricted geometry. The model proteins thaumatin, lysozyme and bacteriorhodopsin demonstrated the feasibility of conducting counter-diffusion equilibration within the new plastic configuration. Crystals of each of these proteins were directly evaluated in situ using synchrotron radiation and their diffraction quality was evaluated without invasive manipulation or cryofreezing. Protein crystals able to produce complete X-ray data sets were used to calculate electron-density maps for structure determination. Fluidic crystallization in the plastic platform was also coupled with a commercialized automated imager and an in situ X-ray scanner that allowed optical and X-ray inspection of crystallization hits. The results demonstrate the feasibility of rapid nanovolume counter-diffusion crystallization experiments without the need for additional instrumentation. Less |Related Solutions: Rock Imager^®

"Protein disorder can plague protein crystallography where the first important goal is to identify conditions that grow stable, ordered, and well diffracting crystals. Initial crystal trials for protease X produced numerous crystals in a myriad of conditions; many of which did not produce well diffracting crystals. Optimizing all of the most obvious parameters: pH, temperature, salt and precipitant concentration continued to produce poor crystals. The fast growth rate and consistently poor diffraction gave an indication that the protein may have some stability issues that could stem from the choice of storage buffer. Recent studies have shown that incorporating thermal melting (Tm) assays into the crystallization screening experiments completely opens up a greater opportunity to explore the protein?s stability prior to crystal trials. This poster will look at the use of thermal shift and the use of automation to screen, characterize and optimize poorly diffracting crystals to well ordered crystals diffracting to beyond 2 angstrom resolution" Less |Related Solutions: Rock Imager^®

Proteins of the cradle-loop barrel metafold are formed by duplication of a conserved βαβ-element, suggesting a common evolutionary origin from an ancestral group of nucleic acid-binding proteins. The basal fold within this metafold, the RIFT barrel, is also found in a wide range of enzymes, whose homologous relationship with the nucleic acid-binding group is unclear. We have characterized a protein family that is intermediate in sequence and structure between the basal group of cradle-loop barrels and one family of RIFT-barrel enzymes, the riboflavin kinases. We report the structure, substrate-binding mode, and catalytic activity for one of these proteins, Methanocaldococcus jannaschii Mj0056, which is an archaeal riboflavin kinase. Mj0056 is unusual in utilizing CTP rather than ATP as the donor nucleotide, and sequence conservation in the relevant residues suggests that this is a general feature of archaeal riboflavin kinases. Less |Related Solutions: Rock Imager^®

Bacterial over-expression of proteins is a powerful tool to obtain soluble protein amenable to biochemical, biophysical and/or structural characterization. However, it is well established that many recombinant proteins cannot be produced in a soluble form. Several theoretical and empirical methods to improve soluble production have been suggested, although there is to date no universally accepted protocol. This report describes, and quantitatively analyses, a systematic multi-construct approach to obtain soluble protein. Although commonly used in several laboratories, quantitative analyses of the merits of the strategy applied to a larger number of target proteins are missing from the literature. In this study, typically 10 different protein constructs were tested for each targeted domain of nearly 400 human proteins. Overall, soluble expression was obtained for nearly 50% of the human target proteins upon over-expression in Escherichia coli. The chance of obtaining soluble expression was almost doubled using the multi-construct method as compared to more traditional approaches. Soluble protein constructs were subsequently subjected to crystallization trials and the multi-construct approach yielded a more than fourfold increase, from 15 proteins to 65, for the likelihood of obtaining well-diffracting crystals. The results also demonstrate the value of testing multiple constructs in crystallization trials. Finally, a retrospective analysis of gel filtration profiles indicates that these could be used with caution to prioritize protein targets for crystallization trials. Less |Related Solutions: Rock Maker^®

A microfluidic device denoted the Phase Chip has been designed to measure and manipulate the phase diagram of multi-component fluid mixtures. The Phase Chip exploits the permeation of water through poly(dimethylsiloxane) (PDMS) in order to controllably vary the concentration of solutes in aqueous nanoliter volume microdrops stored in wells. The permeation of water in the Phase Chip is modeled using the diffusion equation and good agreement between experiment and theory is obtained. The Phase Chip operates by first creating drops of the water/solute mixture whose composition varies sequentially. Next, drops are transported down channels and guided into storage wells using surface tension forces. Finally, the solute concentration of each stored drop is simultaneously varied and measured. Two applications of the Phase Chip are presented. First, the phase diagram of a polymer/salt mixture is measured on-chip and validated off-chip and second, protein crystallization rates are enhanced through the manipulation of the kinetics of nucleation and growth. Less |Related Solutions: Rock Imager^®

The structure of human inosine triphosphate pyrophosphohydrolase (ITPA) has been determined using diffraction data to 1.6 � resolution. ITPA contributes to the accurate replication of DNA by cleansing cellular dNTP pools of mutagenic nucleotide purine analogs such as dITP or dXTP. A similar high-resolution unpublished structure has been deposited in the Protein Data Bank from a monoclinic and pseudo-merohedrally twinned crystal. Here, cocrystallization of ITPA with a molar ratio of XTP appears to have improved the crystals by eliminating twinning and resulted in an orthorhombic space group. However, there was no evidence for bound XTP in the structure. Comparison with substrate-bound NTPase from a thermophilic organism predicts the movement of residues within helix a1, the loop before a6 and helix a7 to cap off the active site when substrate is bound. Less |Related Solutions: Rock Imager^®

Crystals of the apo-form of the vitamin B12 and colicin transporter, BtuB, that diffract to 1.95 Å have been grown by the membrane-based in meso technique. The structure of the protein differs in several details from that of its counterpart grown by the more traditional, detergent-based (in surfo) method. Some of these differences include i) the five N-terminal residues are resolved in meso, ii) residues 57–62 in the hatch domain and residues 574–581 in loop 21–22 are disordered in meso and are ordered in surfo, iii) residues 278–287 in loop 7–8 are resolved in meso, iv) residues 324–331 in loop 9–10, 396–411 in loop 13–14, 442–458 in loop 15–16 and 526–541 in loop 19–20 have large differences in position between the two crystal forms, as have residues 86–96 in the hatch domain, and v) the conformation of residues 6 and 7 in the Ton box (considered critical to signal transduction and substrate transport) are entirely different in the two structures. Importantly, the in meso orientation of residues 6 and 7 is similar to that of the vitamin B12-charged state. These data suggest that the 'substrate-induced' 180-degree rotation of residues 6 and 7 reported in the literature may not be a unique signaling event. The extent to which these findings agree with structural, dynamic and functional insights gleaned from site-directed spin labeling and electron paramagnetic resonance measurements is evaluated. Packing in in meso-grown crystals is dense and layered, consistent with the current model for crystallogenesis of membrane proteins in lipidic mesophases. Layered packing has been used to locate the transmembrane hydrophobic surface of the protein. Generally, this is consistent with tryptophan, tyrosine, lipid and Cα-B-factor distributions in the protein, and with predictions based on transfer free energy calculations. Less |Related Solutions: Rock Imager^®

The cubic phase or in meso crystallization method is responsible for almost 40 solved integral membrane protein structures. Most of these are small and compact proteins. A model for how crystals form by the in meso method has been proposed that invokes a transition between mesophases. In light of this model, we speculated that a more hydrated and open mesophase, of reduced interfacial curvature, would support facile crystallization of bigger and bulkier proteins. The proposal was explored here by performing crystallization in the presence of additives that swell the cubic phase. The additive concentration inducing swelling, as quantified by small-angle X-ray diffraction, coincided with a "crystallization window" in which two, very different transmembranal proteins produced crystals. That the swollen mesophase can grow structure-grade crystals was proven with one of these, the light-harvesting II complex. In most regards, the structural details of the corresponding complex resembled those of crystals grown by the conventional vapour diffusion method, with some important differences. In particular, packing density in the in meso-grown crystals was dramatically higher, more akin to that seen with water-soluble proteins, which accounts for their enhanced diffracting power. The layered and close in-plane packing observed has been rationalized in a model for nucleation and crystal growth by the in meso method that involves swollen mesophases. These results present a rational case for including mesophase-swelling additives in screens for in meso crystallogenesis. Their use will contribute to broadening the range of membrane proteins that yield to structure determination. Less |Related Solutions: Rock Imager^®

Protein crystallization is a difficult and time-consuming task, because to obtain a
crystal, optimization steps are required almost systematically. A tool that
simplifies the optimization of crystallization conditions, and that can be used by
any crystallographer to design a crystallization plate and to visualize its content,
has become a paramount necessity. A free and open-source application has been
developed to automate this task. It is based on a graphical user interface (GUI)
that allows a personalized crystallization plate to be designed. All data used and
generated are saved in XML documents, which allow reuse of the information.
The steps involved in preparing a crystallization plate and the functions of the
GUI designed to perform these steps are described. Plans for future
development are presented. The program was written in Java. The application
and its documentation are available under CeCILL license, which is a Free
Software license agreement. Less |Related Solutions: Rock Maker^®