Search

A method is presented for measuring anisotropic THz response for small crystals, Crystal Anisotropy Terahertz Microscopy (CATM). Sucrose CATM measurements find the expected anisotropic phonon resonances. CATM measurements of protein crystals find the expected broadband water absorption is suppressed and strong orientation and hydration dependent resonant features.

We have used lysozyme as a test case to illustrate the application of a new method of estimating the intensities of Laue multiples reflection data in protein crystallography. Hen egg-white lysozyme, an enzyme with a single polypeptide chain of 129 amino acids, crystallizes in space group P43212 with cell parametersa=b=79·1 A,c=37·9 A. Laue image plate data were collected using synchrotron radiation on station 9·5 at Daresbury with a total exposure time of 0·95 seconds. The data processed were separated into two data sets comprising the singles and the combined singles and deconvoluted multiples. The method of deconvolution was that of Campbell and Hao (1993), which utilizes the intensity variation of theλ-curve. Electron density maps (2F o−F c) based on the two data sets are then compared. This comparison shows that the deconvoluted multiples do indeed contribute usefully to the continuity of the maps due to the improved completeness of the data. A number of map sections along the polypeptide chain, based on the two Laue data sets, are shown for comparison. These include Arg 5, His 15, Phe 38, Asp 52, Tyr 53, Pro 70, Trp 108 and the four disulphide bridges.

X-ray crystallography provides the majority of our structural biological knowledge at a molecular level and, in terms of pharmaceutical design, is a valuable tool to accelerate discovery. It is the premier technique in the field, but its usefulness is significantly limited by the need to grow well-diffracting crystals. It is for this reason that high-throughput crystallization has become a key technology that has matured over the past 10 years through the field of structural genomics. Areas covered : The authors describe their experiences in high-throughput crystallization screening in the context of structural genomics and the general biomedical community. They focus on the lessons learnt from the operation of a high-throughput crystallization-screening laboratory, which to date has screened over 12,500 biological macromolecules. They also describe the approaches taken to maximize the success while minimizing the effort. Through this, the authors hope that the reader will gain an insight into the efficient design of a laboratory and protocols to accomplish high-throughput crystallization on a single-, multiuser laboratory or industrial scale. Expert opinion : High-throughput crystallization screening is readily available but, despite the power of the crystallographic technique, getting crystals is still not a solved problem. High-throughput approaches can help when used skillfully; however, they still require human input in the detailed analysis and interpretation of results to be more successful.

Structural crystallography and nuclear magnetic resonance (NMR) spectroscopy are the predominant techniques for understanding the biological world on a molecular level. Crystallography is constrained by the ability to form a crystal that diffracts well and NMR is constrained to smaller proteins. Although powerful techniques, they leave many soluble, purified structurally uncharacterized protein samples. Small angle X-ray scattering (SAXS) is a solution technique that provides data on the size and multiple conformations of a sample, and can be used to reconstruct a low-resolution molecular envelope of a macromolecule. In this study, SAXS has been used in a high-throughput manner on a subset of 28 proteins, where structural information is available from crystallographic and/or NMR techniques. These crystallographic and NMR structures were used to validate the accuracy of molecular envelopes reconstructed from SAXS data on a statistical level, to compare and highlight complementary structural information that SAXS provides, and to leverage biological information derived by crystallographers and spectroscopists from their structures. All the ab initio molecular envelopes calculated from the SAXS data agree well with the available structural information. SAXS is a powerful albeit low-resolution technique that can provide additional structural information in a high-throughput and complementary manner to improve the functional interpretation of high-resolution structures.

Observations of crystallization experiments are classified as specific outcomes and integrated through a phase diagram to visualize solubility and thereby direct subsequent experiments. Specific examples are taken from our high-throughput crystallization laboratory which provided a broad scope of data from 20 million crystallization experiments on 12 500 different biological macromolecules. The methods and rationale are broadly and generally applicable in any crystallization laboratory. Through a combination of incomplete factorial sampling of crystallization cocktails, standard outcome classifications, visualization of outcomes as they relate chemically, and application of a simple phase diagram approach, we demonstrate how to logically design subsequent crystallization experiments.

Synchrotron X-radiation (SR) is intense, polychromatic and collimated. It is widely exploited, in macromolecular crystallography, particularly using a monochromatized short wavelength beam. The spectral curve of SR, however, ideally lends itself to use of Laue geometry, i. e. the original diffraction experimental arrangement based on a stationary crystal and a polychromatic X-ray beam. Rapid exposure times and time-resolved crystallography studies, e. g. of enzymes, are now possible. Historical objections to the use of Laue diffraction data, particularly the multiplicity distribution, have been found not to be as limiting as once thought. The credentials of the Laue method have been established through a variety of Laue crystal structure analyses, involving photographic film as detector. Recently a three-dimensional arrangement of films, known as a toast-rack, has been used to alleviate problems with spatially overlapping spots. This paper provides a review of these results and then reports several developments. In particular, one of the first Laue analyses using an image plate as detector, namely of a cobalt substituted concanavalin A crystal, is discussed. Recent experimental developments, also at the Daresbury synchrotron, are then described. First, a large toast-rack has been used to record Laue data from a protein crystal. Secondly, a transmission X-ray mirror has been constructed from thin mylar (1.5 μm) and used to provide a λ max filter instead of using aluminium foils. Thirdly, since the Laue method suffers from poor sampling of the low resolution data, a new method (known as LOT) has been introduced.

Structural biology is key to our understanding of the mechanisms of biological processes. This text describes current methods and future frontiers in crystal growth and use of X-ray and neutron crystallography, in the context of automation of crystallization and generation of synchrotron X-ray and neutron beams.

A program,AutoSherlock, has been developed to present crystallization screening results in terms of chemical space. This facilitates identification of lead conditions, rational interpretation of results and directions for the optimization of crystallization conditions.

As part of a training set for automated image analysis, ∼150 000 images of crystallization experiments from 96 diverse macromolecules have been visually classified within seven categories. Outcomes and trends are analyzed., Structural crystallography aims to provide a three-dimensional representation of macromolecules. Many parts of the multistep process to produce the three-dimensional structural model have been automated, especially through various structural genomics projects. A key step is the production of crystals for diffraction. The target macromolecule is combined with a large and chemically diverse set of cocktails with some leading ideally, but infrequently, to crystallization. A variety of outcomes will be observed during these screening experiments that typically require human interpretation for classification. Human interpretation is neither scalable nor objective, highlighting the need to develop an automatic computer-based image classification. As a first step towards automated image classification, 147 456 images representing crystallization experiments from 96 different macromolecular samples were manually classified. Each image was classified by three experts into seven predefined categories or their combinations. The resulting data where all three observers are in agreement provides one component of a truth set for the development and rigorous testing of automated image-classification systems and provides information about the chemical cocktails used for crystallization. In this paper, the details of this study are presented.

Flash-cooling of macromolecular crystals often compromises diffraction quality by increasing the mosaicity. In some cases, cycling the crystal between low temperature (LT) and room temperature (RT) can reverse this increase in mosaicity. Previous studies of RT/LT cycling have focused on the quality of the crystal as it was repeatedly returned to the LT state. Here, crystal quality is explored not only at LT but also when the crystal is returned to RT. The domain model is used to extract information about crystal order from reflection profiles measured from crystals of Escherichia coli beta-galactosidase at both temperatures. Despite optimization of the cryocooling protocol, the mosaicity increases by about sixfold with cooling and is anisotropic at both temperatures. The mosaicity increase is the consequence of a decrease in domain volume, an increase in the variation of domain cell dimensions and an increase in the angular spread between domains. Upon rewarming, the mosaicity recovers substantially, including the somewhat surprising recovery of domain volume, but incompletely. Over multiple RT/LT cycles disorder in both states increases, which appears to mainly arise from radiation damage, although a contribution from cool-thaw processes cannot be ruled out. The analysis further suggests that LT disorder is governed by variability inherent in the cooling process combined with the overall history of the crystal. In contrast, RT disorder appears to be governed principally by the overall history of the crystal. This suggests that with these particular crystals under the experimental conditions used, particularly at high-intensity synchrotron X-ray sources, RT/LT cycling annealing protocols should involve few cycles so as to limit the hysteresis in both temperature states while taking advantage of the inherent variability in the cooling process that may result in improved crystal order at LT.

An efficient optimization method for the crystallization of biological macromolecules has been developed and tested. This builds on a successful high-throughput technique for the determination of initial crystallization conditions. The optimization method takes an initial condition identified through screening and then varies the concentration of the macromolecule, precipitant, and the growth temperature in a systematic manner. The amount of sample and number of steps is minimized and no biochemical reformulation is required. In the current application a robotic liquid handling system enables high-throughput use, but the technique can easily be adapted in a nonautomated setting. This method has been applied successfully for the rapid optimization of crystallization conditions in nine representative cases.

Lysozyme has been used to investigate the effect of microgravity crystallisation on protein crystal perfection. Crystals were grown in the European Space Agency's Advanced Protein Crystallisation Facility onboard the NASA Space Shuttle. Two missions of differing duration took place, Spacehab-1 and IML-2. The microgravity crystallisation time in each was 7 days and 10 hours, and 12 days and 11 hours respectively. The IML-2 crystals had grown much larger than the Spacehab-1 crystals (2.5 mm versus 0.8 mm at maximum). The earth grown control crystals, in each case, reached a size of 0.8 mm at maximum. The perfection of the crystals was evaluated with collimated, intense, synchrotron radiation. This was done using the Laue method, via the spot size, and by monochromatic rocking widths directly. For the Spacehab-1 crystals spot size measurements were carried out on station 9.5 of the SRS, along with an analysis of intensity to sigma ratio, immediately after the mission. Five months later rocking widths were measured at LURE. The IML-2 crystals were evaluated at the ESRF, on BL3 three months after their return to earth and also a further three months later on the joint Swiss-Norwegian beamline. Both the Spacehab-1 and IML-2 crystals were of exceptional perfection with the crystal mosaicity reaching values as small as 0.0010° and 0.0017° respectively. Earth-grown control crystals had values as small as 0.0032° and 0.007° respectively. There is no evidence of ‘shelf-life’ ageing of the crystals, at least over a period of 6 months, since there is close agreement of the mosaicity values from the Spacehab-1 crystals tested within weeks of that mission and the IML-2 crystals tested 6 months after that mission. The perfect mosaic block size has evidently increased over that realised in the earth-grown controls.

Cryocooling is a technique routinely used to mitigate the effects of secondary radiation damage on macromolecules during X-ray data collection. Energy from the X-ray beam absorbed by the sample raises the temperature of the sample. How large is the temperature increase and does this reduce the effectiveness of cryocooling? Sample heating by the X-ray beam has been measured non-invasively for the first time by means of thermal imaging. Specifically, the temperature rise of 1 mm and 2 mm glass spheres (sample surrogates) exposed to an intense synchrotron X-ray beam and cooled in a laminar flow of nitrogen gas is experimentally measured. For the typical sample sizes, photon energies, fluxes, flux densities and exposure times used for macromolecular crystallographic data collection at third-generation synchrotron radiation sources and with the sample accurately centered in the cryostream, the heating by the X-ray beam is only a few degrees. This is not sufficient to raise the sample above the amorphous-ice/crystalline-ice transition temperature and, if the cryostream cools the sample to 100 K, not even enough to significantly enhance radiation damage from secondary effects.

Neutron diffraction is uniquely sensitive to hydrogen positions and protonation state. In that context structural information from neutron data is complementary to that provided through X-ray diffraction. However, there are practical obstacles to overcome in fully exploiting the potential of neutron diffraction, i.e. low flux and weak scattering. Several approaches are available to overcome these obstacles and we have investigated the simplest: increasing the diffracting volume of the crystals. Volume is a quantifiable metric that is well suited for experimental design and optimization techniques. By using response surface methods we have optimized the xylose isomerase crystal volume, enabling neutron diffraction while we determined the crystallization parameters with a minimum of experiments. Our results suggest a systematic means of enabling neutron diffraction studies for a larger number of samples that require information on hydrogen position and/or protonation state.

The location of hydrogen atoms in enzyme structures can bring critical understanding of catalytic mechanism. However, whilst it is often difficult to determine the position of hydrogen atoms using X-ray crystallography even with subatomic (

Chicken egg-white lysozyme was crystallized from ammonium sulfate over the pH range 4.0-7.8, with protein concentrations from 100 to 150 mg ml(-1). Crystals were obtained by vapor-diffusion or batch-crystallization methods. The protein crystallized in two morphologies with an apparent morphology dependence on temperature and protein concentration. In general, tetragonal crystals could be grown by lowering the protein concentration or temperature. Increasing the temperature or protein concentration resulted in the growth of orthorhombic crystals. Representative crystals of each morphology were selected for X-ray analysis. The tetragonal crystals belonged to the P4(3)2(1)2 space group with crystals grown at pH 4.4 having unit-cell dimensions of a = b = 78.71, c = 38.6 A and diffracting to beyond 2.0 A. The orthorhombic crystals, grown at pH 4.8, were of space group P2(1)2(1)2 and had unit-cell dimensions of a = 30.51, b = 56.51 and c = 73.62 A.

Many bioscience fields employ high-throughput methods to screen multiple biochemical conditions. The analysis of these becomes tedious without a degree of automation. Crystallization, a rate limiting step in biological X-ray crystallography, is one of these fields. Screening of multiple potential crystallization conditions (cocktails) is the most effective method of probing a proteins phase diagram and guiding crystallization but the interpretation of results can be consuming. To aid this empirical approach a cocktail distance coefficient was developed to quantitatively compare macromolecule crystallization conditions and outcome. These coefficients were evaluated against an existing similarity metric developed for crystallization, the C6 metric, using both virtual crystallization screens and by comparison of two related 1,536-cocktail high-throughput crystallization screens. Hierarchical clustering was employed to visualize one of these screens and the crystallization results from an exopolyphosphatase-related protein from Bacteroides fragilis, (BfR192) overlaid on this clustering. This demonstrated a strong correlation between certain chemically related clusters and crystal lead conditions. While this analysis was not used to guide the initial crystallization optimization, it led to the re-evaluation of unexplained peaks in the electron density map of the protein and the insertion and correct placement of a sodium, potassium and phosphate atoms in the structure. With these in place, the resulting structure of the putative active site demonstrated features consistent with active sites of other phosphatases which are involved in binding the phosphoryl moieties of nucleotide triphosphates. The new distance coefficient appears to be robust in this application and coupled with hierarchical clustering and the overlay of crystallization outcome reveals information of biological relevance. While tested with a single example the potential applications appear promising.

Crystals of insulin grown in microgravity on Space Shuttle Mission STS-95 were extremely well ordered and unusually large (many >2 mm). The physical characteristics of six microgravity and six earth-grown crystals were examined by X-ray analysis employing superfine φ slicing and unfocused synchrotron radiation. This experimental setup allowed hundreds of reflections to be precisely examined from each crystal in a short period of time. The microgravity crystals were on average 34 times larger, had sevenfold lower mosaicity, had 54-­fold higher reflection peak heights and diffracted to significantly higher resolution than their earth-grown counterparts. A single mosaic domain model could account for the observed reflection profiles in microgravity crystals, whereas data from earth crystals required a model with multiple mosaic domains. This statistically significant and unbiased characterization indicates that the microgravity environment was useful for the improvement of crystal growth and the resultant diffraction quality in insulin crystals and may be similarly useful for macromolecular crystals in general.

A diffraction geometry utilizing convergent X-rays from a polycapillary optic incident on a stationary crystal is described. A mathematical simulation of the resulting diffraction pattern (in terms of spot shape, position and intensity) is presented along with preliminary experimental results recorded from a lysozyme crystal. The effective source coverage factor is introduced to bring the reflection intensities onto the same scale. The feasibility of its application to macromolecular crystal data collection is discussed.

We introduce a new technique, Crystal Anisotropy Terahertz Microscopy (CATM) which directly measures correlated intra-molecular protein vibrations. The terahertz (THz) frequency range (5-100 cm−1) corresponds to global correlated protein motions proposed to be essential to functional conformational changes [1, 2]. CATM accesses these motions above the relaxational background of the solvent and residue side chain librational motions. We demonstrate narrowband features in the anisotropic absorbance for hen egg-white lysozyme (HEWL) single crystals as well as HEWL with triacetylglucosamine (HEWL-3NAG) inhibitor single crystals. By self-referencing to a single orientation, the absorption spectra were obtained and are free from absorption due to librational motions from biological water and artifacts due to multiple reflections within the crystal. The most prominent features for the HEWL crystals appear at 45 cm−1, 69 cm−1, and 78 cm−1 and the strength of the absorption varies with crystal orientation relative to the THz polarization. Similar anisotropic features appear in molecular mechanics calculations suggesting specific correlated mode identification is possible, giving us direct insight to the role of correlated motions in conformational changes necessary for ligand binding and catalysis. This work supported by NSF MRI∧2 grant DBI295998.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Microgravity offers an environment for protein crystallization where there is an absence of convection and sedimentation. We have investigated the effect of microgravity conditions on the perfection of protein crystals. The quality of crystals for X-ray diffraction studies is characterized by a number of factors, namely size, mosaicity and the resolution limit. By using tetragonal lysozyme crystals as a test case we show, with crystal growth in two separate Space Shuttle missions, that the mosaicity is improved by a factor of three to four over earth-grown ground control values. These microgravity-grown protein crystals are then essentially perfect diffraction gratings. As a result the peak to background of individual X-ray diffraction reflections is enhanced by a similar factor to the reduction in the mosaicity. This then offers a particularly important opportunity for improving the measurement of weak reflections such as occur at high diffraction resolution. These microgravity results set a benchmark for all future microgravity and earth-based protein crystallography procedures.

Crystal mosaicity is determined from the measurement of the reflection angular width and can be used as an indicator of crystal perfection. A new method has been developed that combines the use of unfocused synchrotron radiation, super-fine phi slicing and a CCD area detector to simultaneously measure the mosaicity of hundreds of reflections . The X-ray beam characteristics and Lorentz correction are deconvoluted from the resulting reflection widths to calculate the true crystal mosaicity.

A challenge in neutron diffraction experiments is growing large (greater than 1 cu mm) macromolecule crystals. In taking up this challenge we have used statistical experiment design techniques to quickly identify crystallization conditions under which the largest crystals grow. These techniques provide the maximum information for minimal experimental effort, allowing optimal screening of crystallization variables in a simple experimental matrix, using the minimum amount of sample. Analysis of the results quickly tells the investigator what conditions are the most important for the crystallization. These can then be used to maximize the crystallization results in terms of reducing crystal numbers and providing large crystals of suitable habit. We have used these techniques to grow large crystals of Glucose isomerase. Glucose isomerase is an industrial enzyme used extensively in the food industry for the conversion of glucose to fructose. The aim of this study is the elucidation of the enzymatic mechanism at the molecular level. The accurate determination of hydrogen positions, which is critical for this, is a requirement that neutron diffraction is uniquely suited for. Preliminary neutron diffraction experiments with these crystals conducted at the Institute Laue-Langevin (Grenoble, France) reveal diffraction to beyond 2.5 angstrom. Macromolecular crystal growth is a process involving many parameters, and statistical experimental design is naturally suited to this field. These techniques are sample independent and provide an experimental strategy to maximize crystal volume and habit for neutron diffraction studies.