Three conventional robots were subjected to a crystallization screening test involving 18 proteins from T. thermophilus HB8 using the sitting- and hanging-drop vapour-diffusion and microbatch methods. The number of diffraction-quality crystals and the amount of time required to obtain visible crystals depended greatly on the robots used. The combined use of different robots, especially for protein samples exhibiting low crystallization success rates, significantly increased the chance of obtaining diffraction-quality crystals.
It was essential for the structural genomics of Thermus thermophilus HB8 to efficiently crystallize a number of proteins. To this end, three conventional robots, an HTS-80 (sitting-drop vapour diffusion), a Crystal Finder (hanging-drop vapour diffusion) and a TERA (modified microbatch) robot, were subjected to a crystallization condition screening test involving 18 proteins from T. thermophilus HB8. In addition, a TOPAZ (microfluidic free-interface diffusion) designed specifically for initial screening was also briefly examined. The number of diffraction-quality crystals and the time of appearance of crystals increased in the order HTS-80, Crystal Finder, TERA. With the HTS-80 and Crystal Finder, the time of appearance was short and the rate of salt crystallization was low. With the TERA, the number of diffraction-quality crystals was high, while the time of appearance was long and the rate of salt crystallization was relatively high. For the protein samples exhibiting low crystallization success rates, there were few crystallization conditions that were common to the robots used. In some cases, the success rate depended greatly on the robot used. The TOPAZ showed the shortest time of appearance and the highest success rate, although the crystals obtained were too small for diffraction studies. These results showed that the combined use of different robots significantly increases the chance of obtaining crystals, especially for proteins exhibiting low crystallization success rates. The structures of 360 of 944 purified proteins have been successfully determined through the combined use of an HTS-80 and a TERA.
protein crystallization; crystallization screening; crystallization success rates
A cyclic olefin homopolymer-based microfluidics system has been established for protein crystallization and in situ X-ray diffraction.
Microfluidics is a promising technology for the rapid identification of protein crystallization conditions. However, most of the existing systems utilize silicone elastomers as the chip material which, despite its many benefits, is highly permeable to water vapour. This limits the time available for protein crystallization to less than a week. Here, the use of a cyclic olefin homopolymer-based microfluidics system for protein crystallization and in situ X-ray diffraction is described. Liquid handling in this system is performed in 2 mm thin transparent cards which contain 500 chambers, each with a volume of 320 nl. Microbatch, vapour-diffusion and free-interface diffusion protocols for protein crystallization were implemented and crystals were obtained of a number of proteins, including chicken lysozyme, bovine trypsin, a human p53 protein containing both the DNA-binding and oligomerization domains bound to DNA and a functionally important domain of Arabidopsis Morpheus’ molecule 1 (MOM1). The latter two polypeptides have not been crystallized previously. For X-ray diffraction analysis, either the cards were opened to allow mounting of the crystals on loops or the crystals were exposed to X-rays in situ. For lysozyme, an entire X-ray diffraction data set at 1.5 Å resolution was collected without removing the crystal from the card. Thus, cyclic olefin homopolymer-based microfluidics systems have the potential to further automate protein crystallization and structural genomics efforts.
cyclic olefin homopolymers; microfluidics; crystallization; in situ X-ray diffraction
Single crystals of the holoenzyme (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase with ThDP and Mn2+ as cofactors were obtained by the hanging-drop vapour-diffusion method with 35% ethylene glycol as precipitant. Apoenzyme crystals were obtained by sitting-drop vapour diffusion with 70% MPD.
(1R,6R)-2-Succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase, also called MenD, participates in the menaquinone (vitamin K2) biosynthetic pathway. The enzyme is a part of the superfamily of ThDP-dependent enzymes; however, it is the only enzyme known to catalyze a Stetter-like 1,4-addition of a ThDP adduct to the β-carbon of an unsaturated carboxylate. This is the first reported crystallization of the apoenzyme and holoenzyme forms of MenD. The apoenzyme crystals were obtained by sitting-drop vapour diffusion with 70% MPD. However, the crystals were too small to collect diffraction data and a search for better conditions was not successful. Single crystals of the holoenzyme with ThDP and Mn2+ as cofactors were obtained by the hanging-drop vapour-diffusion method with 35% ethylene glycol as precipitant. Diffraction data were collected on a cryocooled crystal to a resolution of 2.0 Å at BioCARS, Advanced Photon Source (APS), Chicago, IL, USA. The crystal was found to belong to space group P212121, with unit-cell parameters a = 106.86, b = 143.06, c = 156.85 Å, α = β = γ = 90°.
SHCHC synthase; MenD; ThDP-dependent enzymes
The X-CHIP (X-ray Crystallography High-throughput Integrated Platform) is a novel microchip that has been developed to combine multiple steps of the crystallographic pipeline from crystallization to diffraction data collection on a single device to streamline the entire process.
The X-CHIP (X-ray Crystallization High-throughput Integrated Platform) is a novel microchip that has been developed to combine multiple steps of the crystallographic pipeline from crystallization to diffraction data collection on a single device to streamline the entire process. The system has been designed for crystallization condition screening, visual crystal inspection, initial X-ray screening and data collection in a high-throughput fashion. X-ray diffraction data acquisition can be performed directly on-the-chip at room temperature using an in situ approach. The capabilities of the chip eliminate the necessity for manual crystal handling and cryoprotection of crystal samples, while allowing data collection from multiple crystals in the same drop. This technology would be especially beneficial for projects with large volumes of data, such as protein-complex studies and fragment-based screening. The platform employs hydrophilic and hydrophobic concentric ring surfaces on a miniature plate transparent to visible light and X-rays to create a well defined and stable microbatch crystallization environment. The results of crystallization and data-collection experiments demonstrate that high-quality well diffracting crystals can be grown and high-resolution diffraction data sets can be collected using this technology. Furthermore, the quality of a single-wavelength anomalous dispersion data set collected with the X-CHIP at room temperature was sufficient to generate interpretable electron-density maps. This technology is highly resource-efficient owing to the use of nanolitre-scale drop volumes. It does not require any modification for most in-house and synchrotron beamline systems and offers a promising opportunity for full automation of the X-ray structure-determination process.
protein crystallization devices; in situ X-ray analysis; crystallization; crystal visual inspection; diffraction data collection
Traditional macroscale protein crystallization is accomplished non-trivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained. This methodology is time consuming and resource intensive, hindering protein structure determination. Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I (PSI) due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements. Structure determination techniques tailored for these ‘difficult to crystallize’ proteins such as femtosecond nanocrystallography are being developed, yet still need specific crystal characteristics. Here, we demonstrate a simple and robust method to screen protein crystallization conditions at low ionic strength in a microfluidic device. This is realized in one microfluidic experiment using low sample amounts, unlike traditional methods where each solution condition is set up separately. Second harmonic generation microscopy via Second Order Nonlinear Imaging of Chiral Crystals (SONICC) was applied for the detection of nanometer and micrometer sized PSI crystals within microchannels. To develop a crystallization phase diagram, crystals imaged with SONICC at specific channel locations were correlated to protein and salt concentrations determined by numerical simulations of the time-dependent diffusion process along the channel. Our method demonstrated that a portion of the PSI crystallization phase diagram could be reconstructed in excellent agreement with crystallization conditions determined by traditional methods. We postulate that this approach could be utilized to efficiently study and optimize crystallization conditions for a wide range of proteins that are poorly understood to date.
membrane protein; concentration gradients; numerical simulation; SONICC
The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-ray diffraction of protein crystals has been explored.
The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-ray diffraction of protein crystals was explored. These studies included (i) comparison of microcrystal positions in cryoloops as determined by SHG imaging and by X-ray diffraction rastering and (ii) X-ray structure determinations of selected proteins to investigate the potential for laser-induced damage from SHG imaging. In studies using β2 adrenergic receptor membrane-protein crystals prepared in lipidic mesophase, the crystal locations identified by SHG images obtained in transmission mode were found to correlate well with the crystal locations identified by raster scanning using an X-ray minibeam. SHG imaging was found to provide about 2 µm spatial resolution and shorter image-acquisition times. The general insensitivity of SHG images to optical scatter enabled the reliable identification of microcrystals within opaque cryocooled lipidic mesophases that were not identified by conventional bright-field imaging. The potential impact of extended exposure of protein crystals to five times a typical imaging dose from an ultrafast laser source was also assessed. Measurements of myoglobin and thaumatin crystals resulted in no statistically significant differences between structures obtained from diffraction data acquired from exposed and unexposed regions of single crystals. Practical constraints for integrating SHG imaging into an active beamline for routine automated crystal centering are discussed.
second-harmonic generation microscopy; crystal centering; imaging
A preliminary X-ray crystal structural study of a soluble cognate T-cell receptor (TCR) in complex with a pMHC presenting the Melan-A peptide (ELAGIGILTV) is reported. The TCR and pMHC were refolded, purified and mixed together to form complexes, which were crystallized using the sitting-drop vapour-diffusion method. Single TCR–pMHC complex crystals were cryocooled and used for data collection.
Melanocytes are specialized pigmented cells that are found in all healthy skin tissue. In certain individuals, diseased melanocytes can form malignant tumours, melanomas, which cause the majority of skin-cancer-related deaths. The melanoma-associated antigenic peptides are presented on cell surfaces via the class I major histocompatibility complex (MHC). Among the melanoma-associated antigens, the melanoma self-antigen A/melanoma antigen recognized by T cells (Melan-A/MART-1) has attracted attention because of its wide expression in primary and metastatic melanomas. Here, a preliminary X-ray crystal structural study of a soluble cognate T-cell receptor (TCR) in complex with a pMHC presenting the Melan-A peptide (ELAGIGILTV) is reported. The TCR and pMHC were refolded, purified and mixed together to form complexes, which were crystallized using the sitting-drop vapour-diffusion method. Single TCR–pMHC complex crystals were cryocooled and used for data collection. Diffraction data showed that these crystals belonged to space group P41/P43, with unit-cell parameters a = b = 120.4, c = 81.6 Å. A complete data set was collected to 3.1 Å and the structure is currently being analysed.
Melan-A; T-cell receptors; class I MHC; HLA-A2
A high-affinity mutant CD8 (haCD8) has been developed with the aim of developing a therapeutic immunosuppressor. In order to fully understand the nature of the haCD8 interaction, this protein was crystallized using the sitting-drop vapour-diffusion method.
The class I CD8 positive T-cell response is involved in a number of conditions in which artificial down-regulation and control would be therapeutically beneficial. Such conditions include a number of autoimmune diseases and graft rejection in transplant patients. Although the CD8 T-cell response is dominated by the TCR–pMHC interaction, activation of T cells is in most cases also dependent on a number of associated signalling molecules. Previous work has demonstrated the ability of one such molecule (CD8) to act as an antagonist to T-cell activation if added in soluble form. Therefore, a high-affinity mutant CD8 (haCD8) has been developed with the aim of developing a therapeutic immunosuppressor. In order to fully understand the nature of the haCD8 interaction, this protein was crystallized using the sitting-drop vapour-diffusion method. Single haCD8 crystals were cryocooled and used for data collection. These crystals belonged to space group P6422 (assumed by similarity to the wild type), with unit-cell parameters a = 101.08, c = 56.54 Å. V
M calculations indicated one molecule per asymmetric unit. A 2 Å data set was collected and the structure is currently being determined using molecular replacement.
CD8αα; high affinity; structure-based design; immunosuppressors
A high throughput, low volume microfluidic device has been designed to decouple the physical processes of protein crystal nucleation and growth. This device, called the Phase Chip, is constructed out of poly(dimethylsiloxane) (PDMS) elastomer. One of the Phase Chip’s innovations is to exploit surface tension forces to guide each drop to a storage chamber. We demonstrate that nanoliter water-in-oil drops of protein solutions can be rapidly stored in individual wells thereby allowing the screening of 1000 conditions while consuming a total of only 10 μg protein on a 20 cm2 chip. Another significant advance over current microfluidic devices is that each well is in contact with a reservoir via a dialysis membrane through which only water and other low molecular weight organic solvents can pass, but not salt, polymer, or protein. This enables the concentration of all solutes in a solution to be reversibly, rapidly, and precisely varied in contrast to current methods, such as the free interface diffusion or sitting drop methods, which are irreversible. The Phase Chip operates by first optimizing conditions for nucleation by using dialysis to supersaturate the protein solution, which leads to nucleation of many small crystals. Next, conditions are optimized for crystal growth by using dialysis to reduce the protein and precipitant concentrations, which leads small crystals to dissolve while simultaneously causing only the largest ones to grow, ultimately resulting in the transformation of many small, unusable crystals into a few large ones.
microfluidics; PDMS; water permeation; high throughput; protein crystallization; phase diagram; nucleation; growth; Ostwald ripening
The Microcapillary Protein Crystallization System (MPCS) is a new protein-crystallization technology used to generate nanolitre-sized crystallization experiments for crystal screening and optimization. Using the MPCS, diffraction-ready crystals were grown in the plastic MPCS CrystalCard and were used to solve the structure of methionine-R-sulfoxide reductase.
The Microcapillary Protein Crystallization System (MPCS) embodies a new semi-automated plug-based crystallization technology which enables nanolitre-volume screening of crystallization conditions in a plasticware format that allows crystals to be easily removed for traditional cryoprotection and X-ray diffraction data collection. Protein crystals grown in these plastic devices can be directly subjected to in situ X-ray diffraction studies. The MPCS integrates the formulation of crystallization cocktails with the preparation of the crystallization experiments. Within microfluidic Teflon tubing or the microfluidic circuitry of a plastic CrystalCard, ∼10–20 nl volume droplets are generated, each representing a microbatch-style crystallization experiment with a different chemical composition. The entire protein sample is utilized in crystallization experiments. Sparse-matrix screening and chemical gradient screening can be combined in one comprehensive ‘hybrid’ crystallization trial. The technology lends itself well to optimization by high-granularity gradient screening using optimization reagents such as precipitation agents, ligands or cryoprotectants.
protein crystallization; Microcapillary Protein Crystallization System
Electron microscopy of two-dimensional (2D) crystals has demonstrated potential for structure determination of membrane proteins. Technical limitations in large-scale crystallization screens have, however, prevented a major breakthrough in the routine application of this technology. Dialysis is generally used for detergent removal and reconstitution of the protein into a lipid bilayer, and devices for testing numerous conditions in parallel are not readily available. Furthermore, the small size of resulting 2D crystals requires electron microscopy to evaluate the results and automation of the necessary steps is essential to achieve a reasonable throughput. We have designed a crystallization block, using standard microplate dimensions, by which 96 unique samples can be dialyzed simultaneously against 96 different buffers and have demonstrated that the rate of detergent dialysis is comparable to those obtained with conventional dialysis devices. A liquid-handling robot was employed to set up 2D crystallization trials with the membrane proteins CopA from Archaeoglobus fulgidus and light-harvesting complex II (LH2) from Rhodobacter sphaeroides. For CopA, 1-week of dialysis yielded tubular crystals and, for LH2, large and well-ordered vesicular 2D crystals were obtained after 24-hours, illustrating the feasibility of this approach. Combined with a high-throughput procedure for preparation of EM-grids and automation of the subsequent negative staining step, the crystallization block offers a novel pipeline that promises to speed up considerably large-scale screening of 2D crystallization and to increase the likelihood of producing well-ordered crystals for analysis by electron crystallography.
Two-dimensional (2D) crystals; membrane proteins; electron crystallography; high-throughput screening; membrane protein reconstitution; negative staining; 96-well format; crystallization block; dialysis block
This paper reports a method for fabricating multilayer microfluidic protein crystallization platforms using different materials to achieve X-ray transparency and compatibility with crystallization reagents. To validate this approach, three soluble proteins, lysozyme, thaumatin, and ribonuclease A were crystallized on-chip, followed by on-chip diffraction data collection. We also report a chip with an array of wells for screening different conditions that consume a minimal amount of protein solution as compared to traditional screening methods. A large number of high quality isomorphous protein crystals can be grown in the wells, after which slices of X-ray data can be collected from many crystals still residing within the wells. Complete protein structures can be obtained by merging these slices of data followed by further processing with crystallography software. This approach of using an x-ray transparent chip for screening, crystal growth, and X-ray data collection enables room temperature data collection from many crystals mounted in parallel, which thus eliminates crystal handling and minimizes radiation damage to the crystals.
X-ray transparency; microfabrication; protein crystallization; structure determination; Crystallography
A thermostable manganese(II)-dependent 2,3-dihydroxybiphenyl-1,2-dioxygenase derived from Bacillus sp. JF8 was crystallized in two forms using the sitting-drop vapour-diffusion method. Both crystals diffracted to approximately 1.3 Å resolution.
A thermostable manganese(II)-dependent 2,3-dihydroxybiphenyl-1,2-dioxygenase derived from Bacillus sp. JF8 was crystallized. The initial screening for crystallization was performed by the sitting-drop vapour-diffusion method using a crystallization robot, resulting in the growth of two crystal forms. The first crystal belonged to space group P1, with unit-cell parameters a = 62.7, b = 71.4, c = 93.6 Å, α = 71.2, β = 81.0, γ = 64.0°, and diffracted to 1.3 Å resolution. The second crystal belonged to space group I222, with unit-cell parameters a = 74.2, b = 90.8, c = 104.3 Å, and diffracted to 1.3 Å resolution. Molecular-replacement trials using homoprotocatechuate 2,3-dioxygenase from Arthrobacter globiformis (28% amino-acid sequence identity) as a search model provided a satisfactory solution for both crystal forms.
extradiol dioxygenases; crystallization robots; metalloproteins
The digestive lysozymes 1 and 2 from M. domestica were crystallized by vapour diffusion. The crystallographic data were processed to a maximum resolution of 1.9 Å in both cases.
Lysozymes are mostly known for their defensive role against bacteria, but in several animals lysozymes have a digestive function. Here, the initial crystallographic characterization of two digestive lysozymes from Musca domestica are presented. The proteins were crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium sulfate or PEG/2-propanol as the precipitant. X-ray diffraction data were collected to a maximum resolution of 1.9 Å using synchrotron radiation. The lysozyme 1 and 2 crystals belong to the monoclinic space group P21 (unit-cell parameters a = 36.52, b = 79.44, c = 45.20 Å, β = 102.97°) and the orthorhombic space group P21212 (unit-cell parameters a = 73.90, b = 96.40, c = 33.27 Å), respectively. The crystal structures were solved by molecular replacement and structure refinement is in progress.
digestive lysozymes; Musca domestica
Wild-type and variant crystals of a recombinant enzyme β-d-glucan glucohydrolase from barley (Hordeum vulgare L.) were obtained by macroseeding and cross-seeding with microcrystals obtained from native plant protein. Crystals grew to dimensions of up to 500 × 250 × 375 μm at 277 K in the hanging-drops by vapour-diffusion. Further, the conditions are described that yielded the wild-type crystals with dimensions of 80 × 40 × 60 μm by self-nucleation vapour-diffusion in sitting-drops at 281 K. The wild-type and recombinant crystals prepared by seeding techniques achived full size within 5–14 days, while the wild-type crystals grown by self-nucleation appeared after 30 days and reached their maximum size after another two months. Both the wild-type and recombinant variant crystals, the latter altered in the key catalytic and substrate-binding residues Glu220, Trp434 and Arg158/Glu161 belonged to the P43212 tetragonal space group, i.e., the space group of the native microcrystals was retained in the newly grown recombinant crystals. The crystals diffracted beyond 1.57–1.95 Å and the cell dimensions were between a = b = 99.2–100.8 Å and c = 183.2–183.6 Å. With one molecule in the asymmetric unit, the calculated Matthews coefficients were between 3.4–3.5 Å3·Da−1 and the solvent contents varied between 63.4% and 64.5%. The macroseeding and cross-seeding techniques are advantageous, where a limited amount of variant proteins precludes screening of crystallisation conditions, or where variant proteins could not be crystallized.
macro- and cross-seeding; wild-type and mutant protein; X-ray diffraction
This paper describes two SlipChip-based approaches to protein crystallization: a SlipChip-based free interface diffusion (FID) method and a SlipChip-based composite method that simultaneously performs microbatch and FID crystallization methods in a single device. The FID SlipChip was designed to screen multiple reagents, each at multiple diffusion equilibration times, and was validated by screening conditions for crystallization of two proteins, enoyl-CoA hydratase from Mycobacterium tuberculosis and dihydrofolate reductase/thymidylate synthase from Babesia bovis against 48 different reagents at 5 different equilibration times each, consuming 12 μL of each protein for a total of 480 experiments using three SlipChips. The composite SlipChip was designed to screen multiple reagents, each at multiple mixing ratios and multiple equilibration times, and was validated by screening conditions for crystallization of two proteins, enoyl-CoA hydratase from Mycobacterium tuberculosis and dihydrofolate reductase/thymidylate synthase from Babesia bovis. To prevent cross-contamination while keeping the solution in the neck channels for FID stable, the plates of the SlipChip were etched with a pattern of nanowells. This nanopattern was used to increase the contact angle of aqueous solutions on the surface of the silanized glass. The composite SlipChip increased the number of successful crystallization conditions and identified more conditions for crystallization than separate FID and microbatch screenings. Crystallization experiments were scaled up in well plates using conditions identified during the SlipChip screenings, and X-ray diffraction data were obtained to yield the protein structure of dihydrofolate reductase/thymidylate synthase at 1.95 Å resolution. This free-interface diffusion approach provides a convenient and high-throughput method of setting up gradients in microfluidic devices, and may find additional applications in cell-based assays.
The Microcapillary Protein Crystallization System (MPCS) is used to successfully optimize protein crystals from 28 out of 29 tested proteins. Six protein structures have been determined from diffraction-ready crystals grown inside and harvested directly from the MPCS CrystalCards, which are compatible with the recently commercialized and automated MPCS Plug Maker instrument.
The Microcapillary Protein Crystallization System (MPCS) is a microfluidic, plug-based crystallization technology that generates X-ray diffraction-ready protein crystals in nanolitre volumes. In this study, 28 out of 29 (93%) proteins crystallized by traditional vapor diffusion experiments were successfully crystallized by chemical gradient optimization experiments using the MPCS technology. In total, 90 out of 120 (75%) protein/precipitant combinations leading to initial crystal hits from vapor diffusion experiments were successfully crystallized using MPCS technology. Many of the resulting crystals produced high-quality X-ray diffraction data, and six novel protein structures that were derived from crystals harvested from MPCS CrystalCards are reported.
protein crystals; microfluids; plugs; genomics
Covalent labeling of macromolecules with trace levels (<1%) of a fluorescent dye is proposed as a means to facilitate finding or detecting crystals in crystallization drops. To test the effects of labeled protein concentration on the resulting X-ray diffraction data, experiments were carried out with the model proteins insulin, ribonuclease, lysozyme and thaumatin, which were labeled with the fluorescent dye carboxyrhodamine. All proteins were labeled on their N-terminal amine and lysozyme was also labeled randomly on lysine side chains in a separate series of experiments. Ribonuclease and N-terminal amine-labeled lysozyme crystals were poorly formed at 10% label concentration and these were not used in subsequent diffraction experiments. All model proteins were tested to 5% labeled protein, and thaumatin and randomly labeled lysozyme gave well formed crystals to 10% labeled protein. In all cases tested, the presence of the label was found to not significantly affect the X-ray diffraction data quality obtained. Qualitative visual-inspection experiments over a range of label concentrations indicated that optimum derivatization levels ranged from 0.025–0.05% for insulin to 0.1–0.25% for thaumatin. Light intensity is a simpler search parameter than straight lines and by virtue of being the most densely packed phase, labeled crystals should be the most intense light sources under fluorescent illumination. For both visual and automated methods of crystal detection, label intensity is a simpler and potentially more powerful search parameter. Screening experiments using the proteins canavalin, -lactoglobulins A and B and chymotrypsinogen, all at 0.5% label concentration, demonstrated the utility of this approach to rapidly finding crystals, even when obscured by precipitate. The use of trace-labeled protein is also proposed to be useful for the automated centering of crystals in X-ray beamlines.
The crystallization-phase-diagram-guided method is effective for growing large protein crystals for neutron protein crystallography.
Neutron protein crystallography (NPC) is a powerful tool for determining the hydrogen position and water orientation in proteins, but a much larger protein crystal is needed for NPC than for X-ray crystallography, and thus crystal preparation is a bottleneck. To obtain large protein crystals, it is necessary to know the properties of the target protein in the crystallization solution. Here, a crystal preparation method of fungal cellulase PcCel45A is reported, guided by the phase diagram. Nucleation and precipitation conditions were determined by sitting-drop vapor diffusion. Saturation and unsaturation conditions were evaluated by monitoring crystal dissolution, and a crystallization phase diagram was obtained. To obtain a large crystal, crystallization solution was prepared on a sitting bridge (diameter = 5 mm). Initial crystallization conditions were 40 µl of crystallization solution (40 mg ml−1 protein with 30.5% 3-methyl-1,5-pentanediol in 50 mM tris-HCl pH 8.0) with a 1000 µl reservoir (61% 3-methyl-1,5,-pentanediol in 50 mM tris-HCl pH 8.0) at 293 K. After the first crystal appeared, the concentration of precipitant in the reservoir solution was reduced to 60% to prevent formation of further crystals. Finally, we obtained a crystal of 6 mm3 volume (3 mm × 2 mm × 1 mm), which was suitable for neutron diffraction.
cellulase; neutron protein crystallography; crystallization phase diagram
The prototype of a universal micromanipulation robot for crystal harvesting is presented, and a robotically harvested trypsin crystal yields a high-resolution structure demonstrating the feasibility of robotic protein crystal harvesting.
High-throughput crystallography has reached a level of automation where complete computer-assisted robotic crystallization pipelines are capable of cocktail preparation, crystallization plate setup, and inspection and interpretation of results. While mounting of crystal pins, data collection and structure solution are highly automated, crystal harvesting and cryocooling remain formidable challenges towards full automation. To address the final frontier in achieving fully automated high-throughput crystallography, the prototype of an anthropomorphic six-axis universal micromanipulation robot (UMR) has been designed and tested; this UMR is capable of operator-assisted harvesting and cryoquenching of protein crystals as small as 10 µm from a variety of 96-well plates. The UMR is equipped with a versatile tool exchanger providing full operational flexibility. Trypsin crystals harvested and cryoquenched using the UMR have yielded a 1.5 Å structure demonstrating the feasibility of robotic protein crystal harvesting.
automated crystal harvesting; crystal mounting; cryoprotection; trypsin; protease; benzamidine complex; protamine; intermolecular contacts; crystallization additives
An acoustic high-throughput screening method is described for harvesting protein crystals and combining the protein crystals with chemicals such as a fragment library.
Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s−1) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.
macromolecular crystallography; acoustic droplet ejection; crystal mounting; drug discovery; chemical biology; high-throughput screening
Crystals of T. gondii ENR in complex with NAD+ and triclosan were grown using the hanging-drop vapour-diffusion method with PEG 8000 as precipitant.
The protozoan parasite Toxoplasma gondii is the causative agent of one of the most widespread parasitic infections of man and is a leading cause of congenital neurological birth defects and the third most common cause of food-borne deaths in the United States. Despite this, to date no drugs are available that provide a fully effective treatment. Recently, the antibacterial agent triclosan was shown to inhibit the fatty-acid biosynthesis pathway in T. gondii and to interact with the enoyl reductase (ENR). In order to analyse the potential of triclosan as a lead compound targeting T. gondii ENR and to explore unique features of the apicomplexan enzyme that could be exploited in future drug development, structural studies have been initiated on T. gondii ENR. Crystals of T. gondii ENR in complex with NAD+ and triclosan were grown using the hanging-drop vapour-diffusion method with PEG 8000 as precipitant. The crystals belong to space group P3221, with approximate unit-cell parameters a = 78.1, b = 78.1, c = 188.5 Å, α = β = 90, γ = 120° and a dimer in the asymmetric unit. Test data were collected to beyond 2.6 Å on cryocooled crystals (100 K) using a Rigaku MM007 rotating-anode X-ray source, revealing that the crystals are suitable for a full structural determination.
enoyl reductase; triclosan; T. gondii
The expression and purification of uridine phosphorylase from V. cholerae and the crystallization and preliminary X-ray structure analysis of the complex of this enzyme with thymidine are reported.
A high-resolution structure of the complex of Vibrio cholerae uridine phosphorylase (VchUPh) with its physiological ligand thymidine is important in order to determine the mechanism of the substrate specificity of the enzyme and for the rational design of pharmacological modulators. Here, the expression and purification of VchUPh and the crystallization of its complex with thymidine are reported. Conditions for crystallization were determined with an automated Cartesian Dispensing System using The Classics, MbClass and MbClass II Suites crystallization kits. Crystals of the VchUPh–thymidine complex (of dimensions ∼200–350 µm) were grown by the sitting-drop vapour-diffusion method in ∼7 d at 291 K. The crystallization solution consisted of 1.5 µl VchUPh (15 mg ml−1), 1 µl 0.1 M thymidine and 1.5 µl reservoir solution [15%(w/v) PEG 4000, 0.2 M MgCl2.6H2O in 0.1 M Tris–HCl pH 8.5]. The crystals diffracted to 2.12 Å resolution and belonged to space group P21 (No. 4), with unit-cell parameters a = 91.80, b = 95.91, c = 91.89 Å, β = 119.96°. The Matthews coefficient was calculated as 2.18 Å3 Da−1; the corresponding solvent content was 43.74%.
uridine phosphorylase; Vibrio cholerae; thymidine
The yeast Epsin-1 (ent1) gene has been cloned and expressed in Escherichia coli. The protein product of a construct containing the ENTH-UIM modules was purified to homogeneity and subjected to crystallization screening using the sitting-drop vapour-diffusion method. Refined conditions containing polyethylene glycol 3350 and Tacsimate yielded thin rod-like crystals.
Members of the Epsin protein family regulate the ubiquitin/clathrin-dependent trafficking of transmembrane proteins. The yeast Epsin-1 (ent1) gene was cloned and expressed in Escherichia coli. The protein product of a construct containing the ENTH-UIM modules was purified to homogeneity and subjected to crystallization screening using the sitting-drop vapour-diffusion method. Refined conditions containing polyethylene glycol 3350 and Tacsimate yielded thin rod-like crystals. X-ray analysis revealed that the crystallographic symmetry is primitive orthorhombic, space group P222, with unit-cell parameters a = 32.7, b = 35.5, c = 110.6 Å and a diffraction limit of 2.3 Å. Matthews coefficient calculations suggested that the crystal contained only the ENTH domain. This was corroborated by Coomassie Blue-stained SDS–PAGE analysis of dissolved crystals.
Epsin; ENTH domain; ubiquitylation/clathrin-dependent endocytosis
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 time-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 to the insertion and correct placement of 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, CDcoeff, 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 related to crystallography appear promising and the distance coefficient, clustering, and hierarchal visualization of results undoubtedly have applications in wider fields.