Summary of selected data sets.
High-throughput protein crystallography can be a time consuming and resource intensive endeavor. Although recent years have seen many advances in the field, screening for suitable crystallization conditions using common commercially available platforms still requires considerable amounts of protein and reagents. Furthermore, diffraction quality testing and data collection typically involve physical extraction and cryogenic freezing of the crystal samples, which may have a significant impact on the integrity of the crystal (Garman, 1999). To acquire high-quality diffraction data, both the crystallization conditions and the cryoprotectants must be further optimized. These steps can be time consuming and are often restricted to experienced users (Alcorn &Juers, 2010). In response to these concerns, the last decade has seen a significant surge of developments in crystallography-aimed microtechnology, specifically the use of crystallization chips. So far, the field is dominated by a range of microfluidic devices (Li & Ismagilov, 2010), with one of the most significant differences between them being the type of crystallization technique they employ. Several devices have been developed, and even commercialized (Topaz® crystallizer, Fluidigm Corp., CA, USA; The Crystal Former, Microlytic Inc., MA, USA) that utilize free interface diffusion (FID) (Hansen et al., 2002). Other chips employ nanochannels to create counter-diffusion crystallization (Hasegawa et al., 2007, Ng et al., 2008, Dhouib et al., 2009) or nanodroplets that simulate batch crystallization (Zheng et al., 2003). There are two clear, parallel implications in all these devices. They are all striving to increase efficiency of the hit identification process, and are offering the possibility of
The X-CHIP (Chirgadze, 2009) addresses the same challenges of high-throughput crystallography with an alternative approach, and has a number of unique additional advantages. In contrast to microfluidic chips, the crystallization process takes place on the chip surface, in droplet arrays of aqueous protein and crystallization reagents mixtures under a layer of oil. These microbatch arrays are made possible by altering the chip surface with a unique coating, creating defined areas of varying hydrophobicity. This paper presents the design of the device and accompanying tools for setting up crystallization trials and mounting the chip for data collection, as well as the important benefits, limitations and implications that are inherent to this platform. It also describes proof-of-concept experiments in which this technology was utilized for crystal growth, visual inspection, X-ray diffraction data collection and structure determination of two native and one selenomethionine-labeled protein targets. The presented results show that large, well-diffracting crystals can be grown and high-quality data sets sufficient for structure determination can be collected on a home as well as a synchrotron X-ray source.
2. X-CHIP design and application
The principal idea behind the X-CHIP was to create a platform that presents an alternative to the conventional crystallographic pipeline by consolidating the processes of crystallization condition screening, crystal inspection and data collection onto one device, streamlining the entire process and eliminating crystal handling and arduous cryogenic techniques (Fig. 1).
The chip is made from a material chosen for its visual light transparency and relatively low absorption of X-ray radiation. An X-CHIP with a thickness of 0.375 mm absorbed approximately thirty percent of the X-ray intensity of the primary synchrotron beam that was attenuated by 1,800-2,000 times to avoid excessive radiation damage to crystals during data acquisition. Designed to be compatible with most standard goniometers, the device inserts into a chip-base (possessing a machined slit and locking screw) for support and simple mounting (Fig. 1). A plastic receptacle holds multiple chips mounted on bases, providing rigidity for set up, storage and visual inspection of the crystallization drops and can be covered with a special lid to prevent dust contamination. The chip, along with supporting tools, is shown in Fig. 2.
The described system applies principles of the microbatch crystallization method, the high effectiveness and unique benefits of which have been described elsewhere (D'Arcy et al., 1996, Chayen, 1998, D'Arcy et al., 2003). On the surface of the chip, circular hydrophilic areas are inscribed in hydrophobic annuli in ordered arrays (Fig. 2a, 2
3. Materials and methods
Previously investigated targets, the protein kinase domain of human Ephrin Receptor Tyrosine Kinase A3 (EphA3) (Davis et al., 2008) and the
In-house data sets were collected on a
Two important aspects of the described system were investigated throughout this study; the capacity of the chip to produce diffraction quality crystals and the feasibility of diffraction data acquisition (
Proof-of-concept experiments for on-the-chip data collection were carried out on the rotating anode source and the synchrotron beamline. The initial data collection trials on the in-house X-ray source led to the acquisition of a complete EphA3 data set. While the experiment was conducted at room temperature, diffraction data could still be obtained with sufficient completeness, even for crystals of such low symmetry space group as P21 (Table 1). At the synchrotron beamline, data sets for EphA3, PA0269 and a PA0269 selenomethionine derivative (SAD) were collected. The high sensitivity and ultrafast readout time of the
|Crystal mount method||X-CHIP||X-CHIP||X-CHIP||X-CHIP|
|Sample Temperature (K)||295||295||295||295|
|X-ray Source||Rigaku FR-E||ID-17 APS||ID-17 APS||ID-17 APS|
|Detector||R-AXIS HTC||Pilatus 6M||Pilatus 6M||Pilatus 6M|
|High resolution shell (Å)||(2.10-2.00)||(2.05-1.95)||(2.05-1.95)||(2.05-1.95)|
|Data Collection Time (min)||100||5.3||3.0||3.3|
|Mosaic spread (o)||0.100||0.360||0.046||0.160|
|<I/σ(I)"/>||4.5 (2.2)||7.5 (2.7)||12.1 (2.7)||20.8 (3.9)|
|Rmerge (%)||8.8 (34.6)||10.9 (34.6)||8.2 (47.5)||6.9 (50.2)|
For crystallization of EphA3 and PA0269, paraffin oil was used to coat the crystallization drops after protein and precipitant solution had been dispensed. Other oils have been explored, such as Hampton's Al's Oil (50/50 paraffin/silicon oil mixture), silicon oil and a 50/50 mix of paratone/paraffin oils. Higher viscosity oils (paraffin, paratone/paraffin) performed better on the X-CHIP by being highly restricted to the hydrophobic ring boundaries. The thinner silicon oil was found to flow outside of these boundaries causing drop merging. Al's oil required more careful application compared to higher viscosity oils, but proved to stay within the hydrophobic boundaries. Crystallization conditions containing ethanol, 2-methyl-2,4-pentanediol (MPD) and detergents were also tested on the X-CHIP. Ethanol tolerance was tested with a 5-30% gradient using paraffin oil as a cover. The phase separation within the crystallization drops remained intact for the entire gradientrange. Crystallization drops containing MPD in combination with different oils tolerated up to 8% before they began to disperse beyond the hydrophobic area the hydrophobic area. While this can exclude some MPD based conditions from being used on the X-CHIP, the impact on the overall versatility is low since most commercially available initial screens from Hampton Research and Emerald Biosystems (e.g. Wizard I&II, Index, Crystal) only have an average of 5-6% total conditions containing MPD. Detergent tolerance was tested with n-dodecyl-N,N-dimethylamine-N-oxide (DDAO) and n-octyl-β-D-glucoside in combination with a paraffin oil covering. Separation between the phases remained intact with the 0.05% n-octyl-b-D-glucoside condition but, DDAO was not tolerated even at very low concentrations.
The series of initial experiments on the X-CHIP crystallization platform described above demonstrated the chip’s applicability for high-throughput protein crystallography and provided insight into the benefits and limitations of this system. Crystallization using the microbatch method on the chip was shown to be suitable for crystal growth and also offered additional benefits. Oil covered drops evaporate very slowly (days to weeks), simplifying both manual and automated set up. Furthermore, changing the composition of the top oil layer with various oil mixtures makes it possible to vary the rate of water evaporation over a wide range, adding another favorable dimension to crystallization screening (D'Arcy et al., 2004). Inherently, the system is economical since crystallization hit determination and optimization trials require up to five times less volume of protein sample and five hundred times less reagent solution than standard vapor diffusion methods. Theoretically, the volumes can be decreased even further by incorporatingrobotic liquid handling systems, but are currently limited by the accuracy of manual dispensing. In addition, the simplicity of the device results in low manufacturing costs and the platform design eliminates the time and expenses associated with cryogenic techniques. The small size of the chip offers more convenient and faster visual inspection, as all the crystallization drops can be viewed under a microscope simultaneously. Furthermore, the system design provides a non-invasive means of diffraction testing and screening, as the developed device can be mounted on most in-house and synchrotron beamline data acquisition systems without any modification of the chip or adjustments to that system. These capabilities of the X-CHIP make it a potentially useful platform for high-throughput initiatives such as fragment-based screening by co-crystallization.
The X-CHIP system has the potential to completely remove the “user factor” between crystal growth and X-ray diffraction data collection, eliminating crystal manipulation. The feasibility of
Current developments on the project are aimed at scaling down the drop volumes of the X-CHIP system. Attempting to do so using manual set up has proven to be challenging, but application of a liquid handling robotics system can address this issue. The
From the initial studies of the device it is evident that not only does the X-CHIP have the potential to increase efficiency and offer on-the-chip
For providing technical support and access to the in-house and synchrotron X-ray sources we are thankful to Aiping Dong of the Toronto Structural Genomics Consortium and the IMCA-CAT staff at the APS, respectively. We also extend our gratitude to Dr. Tara Davis for providing bacterial cell cultures for purification of EphA3, Kathy Jones for her help with initial crystallization experiments, Joe Miller for help with business development and Jason C. Ellis for machining accessory items. Use of the IMCA-CAT beamline 17-ID at the Advanced Photon Source was supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with the Hauptman-Woodward Medical Research Institute. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, contract No. DE-AC02-06CH11357.
Alcorn T. Juers D. H. 2010ActaCrystallogr D BiolCrystallogr D 66 366 373
Chayen N. E. 1998Acta Crystallogr D Biol Crystallogr 54 8 15
Chirgadze N. Y. et al. 2009Patent Application Number WO 2009/073972 A1
D’Arcy A. Elmore C. Stihle M. Johnston J. E. 1996Journal of Crystal Growth 168 175 180
D’Arcy A. Mac Sweeney. A. Stihle M. Haber A. 2003Acta Crystallogr D Biol Crystallogr 59 396 399
D’Arcy A. Sweeney A. M. Haber A. 2004Methods 34 323 328
Davis T. L. Walker J. R. Loppnau P. Butler-Cole C. Allali-Hassani A. Dhe-Paganon S. 2008Structure (London, England : 1993) 16, 873-884.
Dhouib K. Khan Malek. C. Pfleging W. Gauthier-Manuel B. Duffait R. Thuillier G. Ferrigno R. Jacquamet L. Ohana J. Ferrer J. L. Theobald-Dietrich A. Giege R. Lorber B. Sauter C. 2009Lab Chip 9 1412 1421
Garman E. 1999Acta Crystallogr D Biol Crystallogr 55 1641 1653
Hansen C. L. Classen S. Berger J. M. Quake S. R. 2006J Am Chem Soc 128 3142 3143
Hansen C. L. Skordalakes E. Berger J. M. Quake S. R. 2002Proc Natl Acad Sci U S A 99 16531 16536
Hasegawa T. Hamada K. Sato M. Motohara M. Sano S. Kobayashi T. Tanaka T. Katsube Y. 2007Presented at 24th European Crystallographic Meeting, Marrakech, Morocco.
Mc Grath T. E. Battaile K. Kisselman G. Romanov V. Wu-Brown J. Virag C. Ng I. Kimber M. Edwards A. M. Pai E. F. Chirgadze N. Y. 2007O4DProtein Data Bank
Li L. Ismagilov R. F. 2010Annu Rev Biophys 39 139 158
May A. Fowler B. Frankel K. A. Meigs G. Holton J. M. 2008Acta Cryst A64, C 133 134
Ng J. D. Clark P. J. Stevens R. C. Kuhn P. 2008Acta Crystallogr D Biol Crystallogr 64 189 197
Zheng B. Roach L. S. Ismagilov R. F. 2003J Am Chem Soc 125 11170 11171
Zheng B. Tice J. D. Roach L. S. Ismagilov R. F. 2004Angew Chem Int Ed Engl 43 2508 2511