Towards User Operations of SONICC at GM/CA@APS

Second order nonlinear optical imaging of chiral crystals (SONICC), based on femtosecond laser scanning microscopy, has been implemented at GM/CA@APS undulator beamline 23ID-B for rapid protein crystal localization and centering. The technique is based on infrared laser light impinging on non-centrosymmetric crystals of proteins, which selectively may yield a frequency-doubled, visible signal generated by the anharmonic response of the electron cloud of the protein in response to the laser field. One aim of this method is to locate small crystals grown in opaque crystallization media for centering in X-ray beams of only a few microns or less in cross-section [1]. The optical system implemented at the beamline includes `trans' and `epi' detection of Second Harmonic Generation (SHG) signals [2]. In addition, scanning visible laser light across the sample and detecting two-photon excited UV fluorescence (TPE-UVF) provides complementary contrast based on the native fluorescence of proteins. An update on progress towards offering a user-friendly system to users will be provided. Different factors that influence imaging signals and the practice of successfully locating and accurately positioning a crystal via SONICC will be discussed.

Vanin-1- a human ectoenzyme involved in inflammation, metabolism and disease

Inflammation is steadily gaining recognition as contributing to different disease states, e.g. cancer, immune dysfunction and cardiovascular disease. Vanin-1, a GPI-anchored, developmentally regulated member of the nitrilase family, sits at the intersection of many known pathways of inflammation. Three members of the vanin family of enzymes in humans have been described, with vanin-1 and vanin-2 having confirmed enzymatic activity. A known substrate is pantetheine which is hydrolyzed to give vitamin B5 and cysteamine. One function of these enzymes is in pantothenate recycling, and therefore they are involved in the Coenzyme A cycle and metabolism. Mouse knockout studies have shown that Vnn1(-/-) mice are resistant to oxidative stress, intestinal inflammation and colitis. Epidemiological studies have shown that mutations in vanin-1 in humans are associated with child obesity1; other studies show an association with cholesterol homeostasis and cardiovascular disease. Importantly, the metabolic pathways of lipid metabolism and inflammation are interconnected2 whereby impairment of lipid metabolism leads to inflammation and inflammation leads to impairment of lipid metabolism. We recently solved the structure of a soluble form of human vanin-1 using a single heavy atom derivative and anomalous scattering to 2.3 Å resolution. It has two domains: a predicted nitrilase domain and a cap domain which has no known sequence homology to any other structural domain. We also have structures with inhibitors bound and have performed mutational studies to determine the function of the cap domain and affirm the catalytic residues in the catalytic site. Structural studies have been complemented by enzymatic assays showing various levels of activity for the mutant and wild type enzymes. These data will be fundamental in characterizing vanin-1 in different disease states.

PPM Detection Limits in PXRD by Integrating Nonlinear Optics and Synchrotron XRD

SHG microscopy allows rapid and selective identification of trace chiral crystals within amorphous media, enabling targeted XRD using a 5-10 micrometer diameter "minibeam". The sensitivity of PXRD is increased substantially by reducing the background scattering contributions of amorphous material otherwise encountered with a larger beam. In addition, performing diffraction only at the locations most likely to produce diffraction greatly reduced the overall beam-time required to perform the PXRD analyses. Integration of the SHG microscope directly into a synchrotron X-ray beamline at Argonne National Laboratory recovered high spatial registry between the regions of interest identified by SHG for positioning within the X-ray beam. Using this approach, diffraction was performed on individual griseofulvin nanocrystals suspended within an amorphous polymer, corresponding to a total of ~20 fg of total crystalline material. Additional measurements for ritonavir in hydroxypropylmethylcellulose (HPMC) were also performed, in which a bulk API concentration of 100 ppm produced diffraction peaks with a signal to noise ratio of >3000. Among other applications, sensitive detection of trace crystallinity can inform the design of amorphous formulations, in which the bioavailability of active pharmaceutical ingredients (APIs) is enhanced by maintaining them in an amorphous state. However, the long-term stability of a final dosage form can be negatively impacted by spontaneous transitioning to the typically more stable crystalline forms of the APIs, such that extensive quantitative characterization of the crystallization behaviors of amorphous formulations is routinely performed.

Protein Crystal Staining for Second Harmonic Generation Imaging

Recently, second harmonic generation (SHG) microscopy has become a useful tool in the field of structural biology for the detection of protein crystals. SHG, or the frequency doubling of light, is a process specific to crystalline media lacking inversion centers. Through theoretical models and experimental data, it is estimated that ~84% of the known protein crystal structures give detectable SHG signal using current SHG microscopy instrumentation. Extending this coverage could be extremely useful to structural biologists who routinely screen entire 96 well plates, with hundreds of crystallization conditions, in search of diffraction-quality protein crystals. A series of SHG active dyes, including Malachite Green (MG) and trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide (DMI) were investigated to assess their ability to enhance the nonlinear optical (NLO) response across a broad range of protein crystals with varying degrees of inherent SHG activity. MG and DMI were shown to enhance the SHG activity of tetragonal (P43212) lysozyme crystals, a protein that typically generates little to no SHG signal. SHG enhancements for lysozyme of approximately 16000x and 20x were achieved by intercalation of MG and DMI, respectively. These results are consistent with predictions based on the differences in symmetry and structure for the two dyes. The kinetics of the dye intercalation and uptake were investigated with SHG time-lapse images taken of a lysozyme crystal after the addition of MG dye into the crystallization well. Kinetic results indicate that an increase in SHG activity becomes easily noticeable within minutes of exposure to the dyes. These results show a significant progress towards increasing the coverage of SHG microscopy for protein crystal detection.

The $100 million question: Do we need to apply informatics to crystallization?

The PDB is currently growing at a rate of about 9000 structures annually, and 90% of these have been determined by X-ray diffraction methods. Each structure is the result of one or more crystals. Not every protein crystallises nor do all crystals diffract well enough; it has been estimated that of every 10 proteins that are purified, four will show some sign of crystallisation and one will crystallise robustly enough to obtain a structure1. Using these numbers, and making some educated guesses (for example, that most proteins are tested in 1000 crystallisation trials1) these 8000 structures represent 80,000 purified proteins, and 80,000,000 crystallisation trials which are set up each year. The cost of consumables, chemicals and direct labour to set up those trials varies, but can be estimated to be $0.1- $1, excluding the cost of the protein sample and any automation, suggesting that the structural biology community spends between 10 and 100 million each year on crystallisation. Any tools or insight that we can get from data mining or taking a computational approach to rationalize this process may not only profoundly change structural biology, but will make it much less expensive as well. We will discuss approaches to data mining, data standards, and software tools to enable a more rational approach to the process of crystallogenesis.