The serial millisecond crystallography instrument at the Australian Synchrotron incorporating the "Lipidico" injector.

A serial millisecond crystallography (SMX) facility has recently been implemented at the macromolecular crystallography beamline, MX2 at the Australian Synchrotron. The setup utilizes a combination of an EIGER X 16M detector system and an in-house developed high-viscosity injector, "Lipidico." Lipidico uses a syringe needle to extrude the microcrystal-containing viscous media and it is compatible with commercially available syringes. The combination of sample delivery via protein crystals suspended in a viscous mixture and a millisecond frame rate detector enables high-throughput serial crystallography at the Australian Synchrotron. A hit-finding algorithm, based on the principles of "robust-statistics," is employed to rapidly process the data. Here we present the first SMX experimental results with a detector frame rate of 100 Hz (10 ms exposures) and the Lipidico injector using a mixture of lysozyme microcrystals embedded in high vacuum silicon grease. Details of the experimental setup, sample injector, and data analysis pipeline are designed and developed as part of the Australian Synchrotron SMX instrument and are reviewed here.

Bragg coherent diffraction imaging and metrics for radiation damage in protein micro-crystallography.

The proliferation of extremely intense synchrotron sources has enabled ever higher-resolution structures to be obtained using data collected from smaller and often more imperfect biological crystals (Helliwell, 1984). Synchrotron beamlines now exist that are capable of measuring data from single crystals that are just a few micrometres in size. This provides renewed motivation to study and understand the radiation damage behaviour of small protein crystals. Reciprocal-space mapping and Bragg coherent diffractive imaging experiments have been performed on cryo-cooled microcrystals of hen egg-white lysozyme as they undergo radiation damage. Several well established metrics, such as intensity-loss and lattice expansion, are applied to the diffraction data and the results are compared with several new metrics that can be extracted from the coherent imaging experiments. Individually some of these metrics are inconclusive. However, combining metrics, the results suggest that radiation damage behaviour in protein micro-crystals differs from that of larger protein crystals and may allow them to continue to diffract for longer. A possible mechanism to account for these observations is proposed.

Radiation damage in a micron-sized protein crystal studied via reciprocal space mapping and Bragg coherent diffractive imaging.

For laboratory and synchrotron based X-ray sources, radiation damage has posed a significant barrier to obtaining high-resolution structural data from biological macromolecules. The problem is particularly acute for micron-sized crystals where the weaker signal often necessitates the use of higher intensity beams to obtain the relevant data. Here, we employ a combination of techniques, including Bragg coherent diffractive imaging to characterise the radiation induced damage in a micron-sized protein crystal over time. The approach we adopt here could help screen for potential protein crystal candidates for measurement at X-ray free election laser sources.

A new approach for structure analysis of two-dimensional membrane protein crystals using X-ray powder diffraction data.

The application of powder diffraction methods to problems in structural biology is generally regarded as intractable because of the large number of unresolved, overlapping X-ray reflections. Here, we use information about unit cell lattice parameters, space group transformations, and chemical composition as a priori information in a bootstrap process that resolves the ambiguities associated with overlapping reflections. The measured ratios of reflections that can be resolved experimentally are used to refine the position, the shape, and the orientation of low-resolution molecular structures within the unit cell, in leading to the resolution of the overlapping reflections. The molecular model is then made progressively more sophisticated as additional diffraction information is included in the analysis. We apply our method to the recovery of the structure of the bacteriorhodopsin molecule (bR) to a resolution of 7 Å using experimental data obtained from two-dimensional purple membrane crystals. The approach can be used to determine the structure factors directly or to provide reliable low-resolution phase information that can be refined further by the conventional methods of protein crystallography.

Aldose reductase structures: implications for mechanism and inhibition.

During chronic hyperglycaemia, elevated vascular glucose level causes increased flux through the polyol pathway, which induces functional and morphological changes associated with secondary diabetic complications. Inhibitors of aldose reductase (ARIs) have been widely investigated as potential therapeutic agents, but to date only epalrestat is successfully marketed for treatment of diabetic neuropathy, in Japan. Promising compounds during in vitro studies or in trials with animal models have failed to proceed beyond clinical trials and to everyday use, due to a lack of efficacy or adverse side effects attributed to lack of inhibitor specificity and likely inhibition of the related aldehyde reductase (ALR1). Knowledge of the catalytic mechanism and structures of the current inhibitors complexed with ALR2 are means by which more specific and tightly bound inhibitors can be discovered. This review will provide an overview of the proposed catalytic mechanism and the current state of structure-based drug design.

Sorbitol dehydrogenase: structure, function and ligand design.

Sorbitol dehydrogenase (SDH), a member of the medium-chain dehydrogenase/reductase protein family and the second enzyme of the polyol pathway of glucose metabolism, converts sorbitol to fructose strictly using NAD(+) as coenzyme. SDH is expressed almost ubiquitously in all mammalian tissues. The enzyme has attracted considerable interest due to its implication in the development of diabetic complications and thus its tertiary structure may facilitate the development of drugs for the treatment of diabetes sufferers. Modelling studies suggest that SDH is structurally homologous to mammalian alcohol dehydrogenase with respect to conserved zinc binding motif and a hydrophobic substrate-binding pocket. Recently, the three-dimensional (3-D) structure of a mammalian SDH was solved, and it was found that while the overall 3-D structures of SDH and alcohol dehydrogenase are similar, the zinc coordination in the active sites of the two enzymes is different. The available structural and biochemical information of SDH are currently being utilized in a structure-based approach to develop drugs for the treatment or prevention of the complications of diabetes. This review provides an overview of the recent advances in the structure, function and drug development fields of sorbitol dehydrogenase.

Modelling studies of the active site of human sorbitol dehydrogenase: an approach to structure-based inhibitor design of the enzyme.

The program GRID was used to design novel potential inhibitors of human sorbitol dehydrogenase based on a model of the holoenzyme in complex with the inhibitor WAY135 706. Replacement of the methyl hydroxyl group of the inhibitor with methyl phosphate and methyl carboxylate functional groups increased the net binding energy of the complex by 2.0- and 1.7-fold, respectively. This study may be useful in the development of potent and more specific inhibitors of the enzyme.

Modelling studies on the binding of substrate and inhibitor to the active site of human sorbitol dehydrogenase.

This study reports a molecular modelling investigation of human sorbitol dehydrogenase complexed with the substrate sorbitol and the inhibitor WAY135 706 based on the structures of human beta3 alcohol dehydrogenase, human sigma alcohol dehydrogenase and horse liver alcohol dehydrogenase. The tertiary structure of human beta3 alcohol dehydrogenase was used as a template for the construction of the model. The rms positional deviation between the main-chain atoms of the initial and final models of sorbitol dehydrogenase is 1.37 A. Similar residue interactions exist between sorbitol dehydrogenase and both sorbitol and inhibitor. Binding of sorbitol in the substrate-binding site results in interactions with Lys-294, Tyr-50, His-69, Glu-150, and NAD+ while WAY135 706 interacts with Ser-46, Lys-294 and Phe-59. The enzyme-inhibitor interactions revealed by this study will be useful in the design of more specific inhibitors.