MICROFLUIDIC DEVICES AND METHODS FOR PROTEIN CRYSTALLIZATION AND IN SITU X-RAY DIFFRACTION

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Patent Application

Serial No. 60/061,218 filed on June 13, 2008 the contents of which are incorporated herein by reference in their entirety.

FIELD OF THE DISCLOSURE The present disclosure relates to the field of micro fluidic circuits for chemical, biological, and biochemical processes or reactions. In particular, the present disclosure is directed to adapting a microfluidic device for protein crystallization and in situ X-ray diffraction crystal screening.

BACKGROUND OF THE DISCLOSURE

In recent years, the pharmaceutical, biotechnology, chemical and related industries have increasingly adopted devices containing micro-chambers and channel structures for performing various reactions and analyses. These devices, commonly referred to as microfluidic devices, allow a reduction in volume of the reagents and sample required to perform an assay. They also enable a large number of reactions without human intervention, either in parallel or in serially, in a very predictable and reproducible way. Microfluidic devices are therefore promising devices to realize a Micro Total Analysis System (micro-TAS), definition that characterizes miniaturized devices that have the functionality of a conventional laboratory.

In general, all attempts at micro-TAS devices can be characterized in two ways: according to the forces responsible for the fluid transport and according to the mechanism used to direct the flow of fluids. The former are referred to as motors. The latter are referred to as valves, and constitute logic or analogue actuators, essential for a number of basic operations such as volumetric quantitation of fluids, mixing of fluids, connecting a set of fluid inlets to a set of

fluid outputs, sealing containers (to gas or to liquids passage according to the application) in a sufficiently tight manner to allow fluid storage, regulating the fluid flow speed. A combination of valves and motors on a microfluidic network, complemented by input means to load the devices, and readout means to measure the outcome of the analysis, make a micro-TAS possible and useful. With increasing performances and miniaturization of the devices, the need for a reliable and adaptable interface to the macroscopic world becomes a requirement to allow users to exploit the functionality of these systems, both for research and commercial applications. Centripetal devices are a specific class of microfluidic devices, where the micro-fluidic devices are spun around a rotation axis in such a way that the centripetal acceleration generates an apparent centrifugal force on the microfluidic device itself, and on any fluid contained within the microfluidic device. The centrifugal force acts as a motor, in the radial but also in the tangential direction if the angular momentum varies. This force, however, is applied at the same time to any material contained in the microfluidic device, including the fluids that are contained in the inlets. In most centripetal microfluidic devices, like for example those developed by Gyros AB, Tecan AG, Burstein Technologies Inc. for example, micro-fluidic devices have the shape of disks, and the rotation axis is perpendicular to the main faces and passing through the centre of the disk. The centrifugal force, therefore, is also parallel to the surface of the disk: it is evident that non-sealed inputs manufactured on the surface require a very specific shape in order to prevent overspill of the fluid out of the inlet aperture.

Microfluidic devices are potentially a promising technology platform for various biochemical research such as the rapid identification of protein crystallization conditions, however, it use for such is not without technical problems. Most of the existing systems utilize silicone elastomers as the chip material, which, despite its many benefits, is unfortunately highly permeable to water vapors. This disadvantage limits the time available for protein crystallization to less than a week.

Identifying conditions for protein crystallization is a labor-intensive process. First, proteins are mixed with various precipitants under conditions favoring crystallization; possible hits are identified and examined for X-ray diffraction. Automating this process would allow protein structures to be understood in a more rapid and cost-effective manner.

Crystallization of proteins and determination of their three-dimensional structure provides biological information that is often critical to understanding their function. Indeed, it has been proposed that the three dimensional structures of all proteins should be solved and for these efforts the term "structural genomics" has been used. So far, these efforts have met with mixed success (Service, Science 298, 948-950 2002; Chandonia and Brenner, Science 311, 347- 351, 2006), in part because protein crystallization is a tedious and time-consuming process that is not easily amenable to automation. Nevertheless, progress towards automation has been made and currently many crystallographers rely on some level of automation for their daily experiments.

Perhaps, the most widely used systems for automating protein crystallization are pipetting/robotic systems that simply recapitulate the steps performed by humans (Hui and Edwards, J. Struct. Biol. 142, 154-161, 2003). One set of pipetting systems prepares crystallization reactions by mixing a precipitant solution with the protein to be crystallized in very small drops (about 200 nanoliters in volume). These drops are placed in chambers containing a much larger volume of precipitant solution. Through vapor diffusion, the volume of the protein drop slowly decreases, leading to protein crystallization. This type of pipetting system has gained acceptance, because vapor diffusion is a well- established method for protein crystallization (Hui and Edwards, 2003; Chayen and Saridakis, Nat. Methods 5, 147-153, 2008) and because it utilizes about ten times less protein than what would be required, if the same reactions were set up manually. However, a disadvantage of the pipetting systems is that the small volume of the protein precipitant drops leads to significant water evaporation before the chambers are sealed. The degree of evaporation can vary from drop to

drop, creating heterogeneity in the experiment, and occurs even when the reactions are prepared in a humidified environment.

Robotic systems have also been developed to identify crystals in protein- precipitant drops; these systems consist of a cabinet, where crystallization plates are stored, a robotic arm and a microscope (Hui and Edwards, 2003). Several images are acquired from each drop (at various focal planes, since the drops are not flat) and then these images are processed by software that attempts to identify protein crystals, which is an unfortunately hard task because the drop geometry leads to poor images. Further, a robotic system has also been developed to position crystallization trays in the path of a synchrotron x-ray beam (Jacquamet et al., Structure 12, 1219-1225, 2004). This system can screen crystals for their ability to diffract x-rays and allows some crystal parameters, such as space group and cell size, to be determined without manual handling of the crystals. As an alternative to the systems described above, and an attempt to address their shortcomings, efforts have been made to use microfluidic devices for protein crystallization. Several such systems have been developed, most of them employing chips made of elastic silicone (Hansen and Quake, Struct. Biol. 13, 538-544, 2003). In one system, the chips contain wells that are connected by a channel; the elastomeric nature of the chips permits the channel to be sealed by applying mechanical pressure on the chip (Hansen et al., 2002 proc. Natl. Acad. Sci. USA 99, 16531- 16536; Hansen et al., Proc. Natl. Acad. Sci. USA 101, 14431-14436, 2004; Hansen et al., J. Am. Chem. Soc. 128, 3142-3143, 2006). In its most simple form, two wells are filled with protein and precipitant solution, respectively; then the pressure on the channel connecting these two wells is released, allowing the contents of the wells to mix by a process called free interface diffusion.

Depending on the viscosity of the liquids, equilibration of the contents of the two connected chambers is achieved in as little as 1 hour. Crystal growth is followed over a period of a few days, but generally for less than a week. The observation time is limited by evaporation of the water through the silicone, which

is intrinsic to the nature of these prior art microfluidic chips, because silicone elastomer is gas-permeable. On the other hand, water evaporation also offers the advantage that it results in higher protein-precipitant concentrations, which may favor crystallization. Another microfluidics system mixes protein and precipitant in nanoliter volume droplets, which form within water-immiscible fluids flowing inside capillary channels (Zheng et al, J. Am. Chem. Soc. 125, 11170-11171 2003; Zheng et al., Angew. Chem. Int. Ed Engl. 43, 2508-2511, 2004; Li et al., Proc. Natl. Acad. Sci. USA 103, 19243-19248, 2006). The droplets initially form in silicone elastomer channels, but are eventually guided into glass or Teflon® capillary tubes, which are then sealed to prevent evaporation. Depending on the nature of the fluid separating the droplets, this system crystallizes proteins by microbatch or vapor diffusion methods. When the oil separating the droplets is impermeable to water, proteins crystallize by the microbatch method. For vapor diffusion, protein-precipitant droplets alternate with droplets containing high concentrations of salt and are separated by a water-permeable oil; this allows for the slow transfer of water from the protein-precipitant droplets to the high salt droplets, resulting in a vapor diffusion effect.

Prior art crystallization methods, such as hanging drop, sitting drop, dialysis and other vapour diffusion methods suffer from the limitation that the material for analysis and the crystallization medium are exposed to the environment for some time. Unfortunately, this causes smaller volumes to be more susceptible to evaporation during the initial creation of the correct mixture and during the initial period after the volume has been set up. Variations in the external environment also can cause significant variations in the production of crystals even if the rate that the samples are made is unchanged.

The use of silicone elastomer is prevalent in microfluidics systems and offers certain advantages, as described above. However, the water vapor permeability of silicone limits its use in cases where protein crystallization requires incubation with precipitant for more than a few days. Thus, microfluidics

systems that utilize vapor impermeable chips could provide an alternative to the silicone elastomer-based systems. A microfluidics system that uses cyclic olefin homopolymer (COP) as the chip material would address the technical shortcomings of prior art micro fluidic devices.

SUMMARY OF THE DISCLOSURE

The present disclosure is directed generally to microfluidic devices and methods with the purpose of interfacing microfluidic devices with dispensing and fluid handling systems to achieve the rapid identification of protein crystallization conditions.

In one illustrative embodiment according to the disclosure a microfluidic device is fabricated with the use of a cyclic olefin homopolymer (COP) creating a microfluidics system adaptable for protein crystallization and in situ X-ray diffraction. In a further illustrative embodiment liquid handling in the microfluidic system according to the disclosure is performed in about 2 mm thin transparent cards, which contain about 500 chambers, each having a volume of about 320 nano liters.

In another illustrative embodiment connectivity between the chambers is controlled by valves that allow specified volumes of liquid to be transferred from one chamber to another. In addition to liquid handling in real time, connections between adjacent chambers can be established either above or below the liquid level to allow vapor or free interface diffusion, respectively. It is contemplated within the scope of the disclosure that communication between chambers can be liquid communications or gaseous communications.

In a further illustrative embodiment the microfluidic card according to the disclosure enables vapor diffusion by having at least two chambers with gaseous communication between them. It is contemplated within the scope of the disclosure that gaseous communication can be pre-conflgured within the card

design or gaseous communication can be established selectively using a laser to pierce a membrane between at least two chambers.

In yet a further illustrative embodiment the microfluidic card according to the disclosure enables liquid diffusion by having at least two chambers with liquid communication between them.

In another illustrative embodiment the microfluidic card according to the disclosure the liquid in which the crystal has grown is defined by a small thickness of plastic on both sides; for in-site diffraction, this provides an acceptable context; for manipulation. It is contemplated within the scope of the disclosure that the microfluidic card according to the disclosure provides an environment shown to be far more robust than sitting-drop or hanging-drop micro- plates, which are extremely sensitive

In a further aspect of the disclosure the microfluidic device according to the disclosure is useful to establish microbatch, vapor diffusion and free interface diffusion protocols for protein crystallization and to obtain crystals for a number of proteins, including but not limited to 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 (MOMl). In another aspect of the disclosure X-ray diffraction analysis can be achieved by either the microfluidic cards being opened to allow mounting of the crystals on loops or the crystals can be exposed to X-rays in situ. It is contemplated within the scope of the invention that one or more chambers can be fabricated having side walls that are removable allowing for the external analysis of protein crystals.

It is contemplated within the scope of the disclosure that cyclic olefin homopolymer-based microfluidics systems have the potential to further automate protein crystallization and structural genomics efforts.

In yet a further aspect of the disclosure programmable sparse matrix screens within transparent microfluidic cards according to the disclosure are

fabricated of cyclic olefin homoPolymer (COP) allowing rapid screening for both protein crystallization conditions and in situ X-ray diffraction.

In a further aspect of the disclosure, microfluidic chambers, within the microfluidic cards according to the disclosure, are connected on the fly, based on a user designed protocol, enabling a wide variety of on-chip assays to be performed unattended, with final volumes in the range of about 100 to about 250 nano liters. It is contemplated within the scope of the disclosure that the microfluidic cards provide unique combinatorial multiplexing capabilities and makes them well suited to serve as a high-throughput screening platform for protein crystallography and the like.

In another aspect of the disclosure the microfluidics cards contain enough volume in each reaction chamber to grow crystals on the order of about 200 to about 300 microns. It is contemplated within the scope of the disclosure that diffraction patterns can be obtained from the crystals without removing them from the screening card, thus drastically reducing hit-to-lead optimization time.

In yet a further illustrative embodiment of the microfluidic cards according to the disclosure the programmability of the fluid mixing in the cards offers a high-level of customization that can be applied to many protocols such as: optimization of conditions with additives, cryo-preservation, co-crystal experiments with ligands, small molecules or the like.

It is also contemplated within the scope of the disclosure that individual cards can be separated and processed independently if required by cutting the film sealing the brick in the direction parallel to the main faces, therefore with the possibility of keeping the card sealed after removal from the brick assembly. In a further aspect of the disclosure protein crystallization of several proteins at about 4o C and at room temperature using microbatch, vapor diffusion and free interface diffusion protocols.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other advantages, objects and features of the disclosure will be apparent through the detailed description of the embodiments and the drawings attached hereto. It is also to be understood that both the foregoing general description and the following detailed description are exemplary and not restrictive of the scope of the disclosure.

Figs. IA, IB, 1C and ID depict an embodiment of a rotor card according to the disclosure, where the inlets are on the small side of the card and the card can be designed to fit into a brick; Fig. 2 depicts a design for input interfaces according to the disclosure, optimized for injection moulding mass-production;

Fig. 3 illustrates another specific embodiment according to the disclosure where the side inputs can be manufactured so that microfluidic structures and inlets on the cards are physically separated during the production of the substrate; Fig. 4 depicts a single card according to the disclosure that is partially sealed by application of a film that prevents the fluid evaporation;

Fig. 5 depicts a single card according to the disclosure;

Fig. 6A is an image of the microfluidics card according to the disclosure (COP Card) wherein samples are loaded on the top of the card and then move through the card by centrifugal force;

Fig. 6B is an image of a cross section of a COP Card illustrating how defined volumes of liquid are "pipetted";

Fig. 6C is an image showing how equal volumes of protein and precipitant were dispensed in one chamber and precipitant only was dispensed in an adjacent chamber;

Fig. 6D is an image showing changes in liquid volume consistent with vapor diffusion after 6 days of incubation of the COP card at room temperature;

Fig. 6E is an image showing free interface diffusion protocol according to the disclosure;

Fig. 7A is an image showing Lysozyme and trypsin crystals formed in COP cards with the microbatch protocol according to the disclosure;

Fig. 7B is an image showing crystallization of human p53/DNA complexes in COP cards using the microbatch and vapor diffusion according to the disclosure;

Fig. 7C is an image showing crystallization of A. thaliana MOMl in COP cards using the microbatch, vapor diffusion and free interface according to the disclosure;

Fig. 8 A is an image showing X-ray diffraction pattern of a p53/DNA crystal exposed to the X-ray beam, while still in the COP card according to the disclosure;

Fig. 8B is an image showing X-ray diffraction patterns of a lysozyme crystal exposed to the X-ray beam, while still in the COP card according to the disclosure; and Fig. 8C shows the lysozyme electron density map contoured at 2 sigma for the 2Fo-Fc map (orange) and at 3 sigma for the Fo-Fc maps (dark blue, positive values; navy blue, negative values).

DETAILED DESCRIPTION OF THE DISCLOSURE The present disclosure provides microfluidic cards that are used within centrifugal rotors and microsystems and in particular nano-scale or meso-scale microfluidic platforms as well as a number of its applications for providing centripetally-motivated fluid micromanipulation. These microfluidic cards are more fully described in PCT application US2005/027867 filed on August 4, 2005, the contents of which are incorporated in its entirety. For the purpose of illustration, the drawings as well as the description will generally refer to centripetal systems. However, the means disclosed in this disclosure are equally applicable in microfluidic components relying on other forces to achieve fluid transport.

For the purposes of this specification, the term "sample" will be understood to encompass any fluid, solution, tissue, cells, proteins, nucleic acids or mixture thereof, either isolated or detected as a constituent of a more complex mixture, or synthesized from precursor species. For the purposes of this specification, the term "in fluid communication" or "fluidly connected" is intended to define components that are operably interconnected to allow fluid flow between components. In illustrative embodiments, the micro-analytical platform comprises microfluidic cards within a rotatable platform, such as a disk, or experimental micro-fluidic chips whereby fluid movement on the chip is motivated by centripetal force upon rotation of the chip and fluid movement on the experimental chip is motivated by pumps.

For the purposes of this specification, the term "biological sample", "sample of interest" or "biological fluid sample" will be understood to mean any biologically-derived analytical sample, including but not limited to blood, plasma, serum, lymph, saliva, tears, cerebrospinal fluid, urine, sweat, plant and vegetable extracts, semen, or any cellular or cellular components of such sample.

For the purposes of this specification, the term "nano-scale" will be understood to mean any volume, able to contain as fluids, with dimensions preferably in the sub-micron to millimetre range. Representative applications of microfluidic cards within a centripetal system (e.g., centrifuge) employ rectangular shaped devices, with the rotation axis positioned outside the device's footprint. For the purpose of illustration, the drawings, as well as the description, will generally refer to such devices. Other shapes other than rectangular shaped devices are contemplated within the scope of the disclosure including but not limited to elliptical and circular devices, irregular surfaces and volumes, and devices for which the rotation axis passes through the body structure, may be beneficial for specific applications.

Turning to Figs. IA and IB a first illustrative embodiment a card 101 according to the disclosure is shown. The card 101 is a substantially planar object formed from a first substrate 102 and a second substrate 106. It is contemplated

within the scope of the disclosure that the card 101 can be also formed from more than two substrates. The substrates 102, 106 can be of any geometric shape. The substrates 102, 106 contain depressions, voids or protrusions that form micro fluidic structures when the substrates are bond together. In a first illustrative embodiment the substrates 102, 106 have a film layer 110 sandwiched between them. The film layer 110 allows for separation of voids within the substrates forming microfluidic circuits that can be placed in fluid communication within each other by perforation of the film layer 110. It is contemplated within the scope of the disclosure that the substrates 102, 106 can be joined within the film layer 110 in between them.

In this first illustrative embodiment the card 101 is substantially rectangular structure having an input end 103, a bottom end 105, a first planar surface 109 and a second planar surface 108. The bottom end 105 has an affixing tab 107 allowing for handling and insertion of the card 101 into a holder or frame. In this illustrative embodiment the input end 103, which is also referred to as a small face, has a plurality of input ports 113. The input ports 113 are in fluid communication with at least one fluid handling microfluidic circuit 115. It is contemplated within the scope of the disclosure that these microfluidic circuits 115 may be composed of a series of valves, chambers, reservoirs, microreactors and microcapillaries. It is also contemplated within the scope of the disclosure that the series of microreactors and microcapillaries are in fluid communication with a detection chamber.

The card 101 has an accessory area 117, which can be used for the purpose of manufacturing, handles, structural supports, precision spacers, purging volumes, bonding areas, identification areas or the like.

The functionality of a specific microfluidic circuit 115 can be configured within the card 101 to perform a desired assay upon a selected sample. It is contemplated within the scope of the disclosure that any microfluidic or fluidic assay known in the art can be configured within the card 101 to achieve a desired functionality. With reference to Fig. 1C a fluidic circuit 121 is shown having a

first state having a reagent contained in a first 120 and second 122 reservoir. With further reference to Fig. ID, the fluidic circuit 121 is shown in a second state after valving within a valving matrix 123 is actuated. It is envisioned that the inventive cards 101 can having a plurality of fluidic circuits 121 that can perform processes in different regions, by actuating the valving matrix 123 as illustrated by the first and second state of the fluidic circuit 121 as depicted in Figs. 1C and ID.

As illustrated in Fig. 1C, a method of joining two fluids in given proportions at a selected time is shown with respect to a first reagent within the first reservoir 120 and a second reagent within the second reservoir 122. According to the disclosure the first and second reagents are transfer in a desired proportion to a mixing chamber 125. The desired proportion of each reagent is delivered to the mixing chamber 125 by actuating the valving matrix 123 as depicted in Fig. ID. These reagents can include but not be limited to the dilution of a reagent into a buffer, the occurrence of a chemical reaction with a given ratio of volumes of reagents, modification of the pH of a solution by addition of an acid or a base, an enzymatic assay where a protein comes into contact with an antibody, or the like.

The fluid handling process starts by the opening of a valve 130 within the valving matrix 123, which could of the type described in the patent application WO04050242A2 ('242 application), wherein the film layer is perforated to actuate a valve. The contents of the '242 application are incorporated herein by reference in their entirety. It is contemplated within the scope of the disclosure that the valving mechanism could also be of different types known in the art such as a mechanical valve or the like. According to the disclosure the reservoirs 120, 122 are positioned onto a different plane with respect to connecting capillaries within the valving matrix 123, and they are separated by means of the film layer 110 that can be perforated at a selected location(s) by irradiation, therefore producing a virtual valve 130 as shown in Fig. ID.

The opening of valves 130, together with the application of a non- equilibrated force onto fluids, allows for the movement of liquids into the mixing

chamber 125. The non-equilibrated force could be generated by means known in the art. In this first illustrative embodiment the non-equilibrated force is achieved by centrifugation so that the liquids are subject to a centripetal acceleration directed towards the bottom of the card 101. According to the disclosure the amount of fluids which are transferred to the mixing chamber 125 is determined by the radial position of valves 130, since only the fluid contained above the corresponding valve 130 is allowed to descend into the mixing chamber 125. The process could be replicated in a plurality of subsequent layers, giving the possibility of successive dilution over various orders of magnitude, mixing two or more type of liquids together, incubating fluids for a given amount of time into the reactors, or even performing a real-time protocol over the matrix layers.

Turning to Fig. 2, a second illustrative embodiment depicting a microfluidic card according to the disclosure is shown. The micro flui die card 210 is comprised of a first substrate 200 and a second substrate 201. The joining of the two substrates 200, 201 forms the microfluidic card 210. The microfluidic card 210 has a bottom face 202, an input face 203, a first planar face (not shown) and a second planar face 207. The input face 203, also known as the small face, of the microfluidic card 201, contains a plurality of input ports 209 in a first input row 211 and a second input row 212. The input face 203 is extruded outside the space confined between the first and second planar faces in order to cause a plurality of microfluidic cards 210 forming a brick having a desired portal interface.

In this illustrative embodiment, the input face 203 contains input ports 209 that have a pitch and opening dimensions of a standard 384 well micro-plate format. It is contemplated within the scope of the disclosure that the input ports 209 can be configured to adapt to any standard laboratory interface. The microfluidic card 210 is suited to manual loading operations, since it is easier to avoid cross-contamination between the inputs ports 209 and to locate the desired input port(s) 209 on the microfluidic card 210. According to the disclosure, inputs ports 209 are manufactured symmetrically on the substrates 200 and 201 forming

the micro fluidic card 210. These substrates 200, 201 are not simply connected, since their inputs are in fluidic communication with the microfluidic components present at the contact surface of substrates 200 and 201, which is also the surface at which substrates 200, 201 are bonded together. Turning to Fig. 3, an example of a device manufactured by bonding simply connected substrates is shown. A first substrate 301 and a second substrate 303 form a microfluidic card 305. Inputs 307 are manufactured as depressions on either substrate 301, 303. These depressions are manufactured by microstructuring means. It is contemplated within the scope of the disclosure that the depressions could also be manufactured by macroscopic means with limited accuracy, for example by milling.

During the manufacturing step, the inputs 307 are not in fluid communication with microfluidic circuits on either on substrate 301 or 303. When the microfluidic card 305 is assembled there is fluidic communication between the microfluidic circuits and the inputs 307. When the two substrates 301, 303 are bond together fluidic communication with the microfluidic structures is established through the substrates 301, 303. Similarly, all other inputs ports 307 can be put in fluidic communication with the microfluidic circuit of the microfluidic card 305. As shown in Fig. 4, a typical requirement of permanent storage applications, like the distribution of a diagnostics assay on a microfluidic device, require reagents to be stored in liquid, solid, encapsulated or lyophilized form inside the microfluidic device. A card 401 according to the disclosure having input ports 401 are subsequently sealed by the use of an impermeable cover 403. The use of the impermeable cover 403 covering inputs ports 402 is done routinely in drugs discovery when using standard micro-plates between the operation of loading reagents and the actual assay. The impermeable cover 403 prevents minute quantities of fluid from evaporating, with the consequence of changing their concentration and therefore modifying the assay conditions.

It is contemplated within the scope of the disclosure that the impermeable cover 403 can be fabricated from polymeric material, natural rubber, or any material having the feature of being inert to liquids used and pierceable for the introduction of liquids, while maintaining gas tightness afterwards to prevent evaporation of store reagents. It is further contemplated within the scope of the disclosure that the impermeable cover 403 can be obtained by application of a laminated film containing metallic and polymeric layers. The metallic layer allows a low permeability to gas and liquids, and the polymeric layer allows for an easy and effective sealing of the store reagents within the card 402. Turning to Figs. 5 A, 5B and 5C, a planar microfluidic card 501 is produced by micro-structuring a facing surface of one, or both, of a first 503 and second 504 facing substrates. Inputs ports 505 are manufactured in one of the two facing substrates 503, 504 and are completely contained inside one or both of the facing substrates 503, 504. The inputs ports 505 have a length inside the substrates 503, 504 that can be decided arbitrarily accordingly to the fluid volumes to be loaded and the pitch between successive input ports 505 can be chosen accordingly to existing standards and specific integration needs. The nominal pitch values of 2.25 mm, 4.5 mm or 9 mm correspond to the 1536, 384 and 96 wells micro-titre plate standards respectively. In this illustrative embodiment, the pitch chosen corresponds to the 1536 micro-titre plate format, with input ports 505 having a square opening.

The substrate 503, 504 with input ports 505 are simply connected. The input ports 505 can be generated by the same mould insert required for the generation of the microstructures forming the microfluidic circuit, or by a second insert (or mould component) sitting on the same side of the microfluidic circuit generating insert. In both cases, removing the piece from the mould is possible without the requirement of movable parts.

Establishment of protein crystallization protocols in COP microfluidics cards.

As shown in Figure 6A, which is an image of a microfluidics card 601 according to the disclosure and as described in Figs 1-5, samples are loaded into inlets 602 on top of the microfluidics card 601. The samples move through the card 601 via centrifugal force 603.

Turning to Fig. 6B, a diagram of a cross section of a microfluidics card

601 according to the disclosure is shown. The micro fluidic card 601 illustrates how defined volumes of liquid are "pipetted". Liquid 605 is contained within a chamber 606 by a thin membrane 607 separating the chambers 606 from vertical channels 608. Openings 609 are made in the thin membrane 607 by an electronic radiation source such as a laser 610. The volume of liquid 611 above the opening

609, moves through the vertical channel 608 to a chamber 606 "below." The volume transferred is determined by the vertical position of the openings 609 in the thin membrane 607.

As shown in Fig. 6C, equal volumes of protein 620 and precipitant 622 were dispensed in a first chamber 624 of the micro fluidic card 601. In a second chamber 626 adjacent to the first chamber 624 of a micro fluidic card 601 a precipitant 622 was dispensed. Then openings 609 were made in the thin membrane above the liquid level to establish connections between the chambers, according to paths 628 depicted within the card 601.

As depicted in Fig. 6D the level of liquid at day 0 630 is indicated by the red lines. The level of liquid in the precipitant chamber 632 increases, while the level of liquid in the protein/precipitant chamber 634 decreases. In this illustrative embodiment, the protein was lysozyme and a crystal 636 was formed within 6 days.

As shown in Fig. 6E, protein 640 and precipitant 642 were dispensed in a first chamber 644 and a second chamber 646 adjacent to each other. Holes were opened in the thin membrane below the liquid level to establish connections between the chambers 644, 646. All images of individual chambers 644, 646 were acquired using a camera built in the microfluidics instrument. Images

showing multiple chambers were assembled from images acquired using an inverted microscope and a low magnification lens (Zeiss, Gottingen, Germany). Protein crystallization in COP cards.

As shown in Fig. 7 A lysozyme 701 and trypsin 702 crystals can be formed in COP cards according to the disclosure with a microbatch protocol. As evident in Fig. 7B crystallization of human p53/DNA complexes in COP cards according to the disclosure using the microbatch and vapor diffusion protocols and three precipitant solutions (index screens #87, #89 and #90). Each condition was performed in triplicate or quadruplicate (numbered 1-3 and 1-4, respectively) and the results are color-coded as follows: protein precipitate, grey; protein crystals, purple; clear solution, white. Examples of crystals that formed by each protocol are shown.

Turning to Fig. 7C crystallization of A. thaliana MOMl in COP cards according to the disclosure using the microbatch, vapor diffusion and free interface diffusion protocols. Each condition was performed in quadruplicate (numbered 1-4) using magnesium formate as the precipitant at the indicated concentrations (M). The results are color-coded, as described for the p53/DNA complexes in (B). For the free interface diffusion protocol, both the protein (left half) and the precipitant (right half) chambers were scored, since over time both chambers will contain both protein and precipitant. Examples of crystals that formed by each protocol are shown. All images were acquired using the camera built in the microfluidics instrument. The width of the chambers is about 750 microns. The detailed compositions of the precipitant solutions are described herein.

Collection of X-ray diffraction datasets from crystals in COP cards.

As shown in Fig 8 A X-ray diffraction pattern of a p53/DNA crystal exposed to the X-ray beam, while still in the COP card. The oscillation range was lo. Turing to Fig. 8B X-ray diffraction patterns of a lysozyme crystal exposed to the X-ray beam, while still in the COP card is shown. Two regions of the

diffraction image are shown, one encompassing a resolution range lower then 3.5 A (left) and the other a region from 1.9-1.6 A (right). The oscillation range was lo. Note that the COP absorbs X-rays in the resolution range between 5.4-5.1 A.

As shown in the table 1 below data collection and refinement statistics from a lysozyme dataset comprised of 45 consecutive frames, each having an oscillation range of lo. R-meas and Rmrgd-F were calculated as described by

Diederichs and Karplus (1997). R factor = (Fobs - Fcalc| / |Fobs|, where Fobs and Fcalc are the observed and calculated structure factors, respectively. Rfree is the R factor calculated using 5% of the reflection data chosen randomly and omitted from the start of refinement. RMS deviations for bonds and angles are the respective root-mean-square deviations from ideal values. Table 1

Observations; 73,037

Unϊ<pe ReSettiøiis: 18,458

Data Coverage {ψ»)ι 91.7

I/Sigi&a: 12.54 Last Shell: 4.09

R~mms (%): 7.9 Last Shell 36.6

8.1 Last Shell 34.3

Water Mβleealess 67

R factor (%); 21.0

Re* (%): 21.9

EMS ϊ>evs: Bonds (Angstroms): 0.004

Angles (degrees): 1.285

As shown in Fig. 8 C the lysozyme electron density map contoured at 2 sigma for the 2Fo-Fc map (orange) and at 3 sigma for the Fo-Fc maps (dark blue, positive values; navy blue, negative values). The map shows the disulfide bond between Cysl33 (C133) and Cys48 (C48) and the side chains of Phe52 (F52) and Trpl41 (W141).

Cards according to the disclosure are advantageously provided having a variety of composition and surface coatings appropriate for a particular application. Card composition will be a function of structural requirements, manufacturing processes, reagent compatibility and chemical resistance properties. In particular, cards may be made from inorganic crystalline or amorphous materials, e.g. silicon, silica, quartz, inert metals, or from organic materials such as plastics, for example, cyclic olefin homopolymer (COP), poly(methylmethacrylate) (PMMA), acetonitrile-butadiene-styrene (ABS), polycarbonate, polyethylene, polystyrene, polyolefins, polypropylene and metallocene. These may be used with unmodified or modified surfaces. In particular fabricating the card from cyclic olefin homopolymer is preferable as it has a low water permeability (0.01%) and significantly low moisture permeability among all plastic polymers

Surface properties of these materials may be modified for specific applications. Surface modification can be achieved by such methods as known in the art including by not limited to silanization, ion implantation and chemical treatment with inert-gas plasmas. It is contemplated within the scope of the disclosure that cards can be made of composites or combinations of these materials, for example, cards manufactured of a polymeric material having embedded therein an optically transparent surface comprising for example a detection chamber of the card.

It is further contemplated within the scope of the disclosure that cards can be fabricated from plastics such as Teflon, polyethylene, polypropylene, methylmethacrylates and polycarbonates, among others, due to their ease of moulding, stamping and milling. It is also contemplated within the scope of the

disclosure that cards can be made of silica, glass, quartz or inert metal. The cards having a fluidic circuit within in one illustrative embodiment can be built by joining using known bonding techniques opposing substrates having complementary micro fluidic circuits etched therein. Cards of the disclosure can be fabricated with injection moulding of optically-clear or opaque adjoining substrates or partially clear or opaque substrates. The cards can be square, rectangular or any geometric form with a thickness approximately comprised between 1 mm and 10 mm. Optical surfaces within the substrates can be used to provide means for detection analysis or other fluidic operations such as laser valving. Layers comprising materials other than polycarbonate can also be incorporated into the cards.

The cards of the disclosure are preferably optically clear, transparent, translucent or opaque and is preferably formed of a material such as cyclic olefin homopolymer (COP) that allows for various spectroscopic analyses (e.g., Raman, UV/IS, IR or x-ray spectroscopy, polarization, fluorescent, and with suitable designs, x-ray diffraction) to be performed in situ.

In order to improve the performance of the device for performing in situ x- ray spectroscopy such as x-ray diffraction and other forms of spectroscopy where an x-ray is caused to traverse the substrate, the number of electrons in the path of the x-ray beam of the material being analyzed should be maximized relative to the number of electrons that is otherwise in the path of the x-ray beam.

The composition of the substrates forming the card depends primarily on the specific application and the requirements of chemical compatibility with the reagents to be used with the card. Electrical layers and corresponding components can be incorporated in cards requiring electric circuits, such as electrophoresis applications and electrically-controlled valves. Control devices, such as integrated circuits, laser diodes, photodiodes and resistive networks that can form selective heating areas or flexible logic structures can be incorporated into appropriately wired areas of the card. Reagents that can be stored dry can be introduced into appropriate open chambers by spraying into reservoirs using

means known in the art during fabrication of the cards. Liquid reagents may also be injected into the appropriate reservoirs, followed by application of a cover layer comprising a thin plastic film that may be utilized for a means of valving within the fluidic circuits within the card. The inventive microfluidic cards may be provided with a multiplicity of components, either fabricated directly onto the substrates forming the card, or placed on the card as prefabricated modules. In addition to the integral fluidic components, certain devices and elements can be located external to the card, optimally positioned on a component of the card, or placed in contact with the card either while rotating within a rotation device or when at rest with a brick formation or with a singular card.

Fluidic components optimally comprising the cards according to the disclosure include but are not limited to detection chambers, reservoirs, valving mechanisms, detectors, sensors, temperature control elements, filters, mixing elements, and control systems.

EXAMPLES

The following examples are provided to illustrate the methods and products of the present disclosure with particular choices for the several components described above. As described above, many variations on these particular examples are possible. These examples are merely illustrative and not limiting of the present disclosure.

Example I Principle of operation of a cyclic olefin homopolymer-based microfluidics device

Most microfluidic devices use either silicone elastomers or rigid COPs as the chip material. The vapor permeability of COPs is several orders of magnitude lower than that of silicone (Mair et al., Lap Chip 6, 1346-1354, 2006), which, in theory, should make COPs better suited for traditional methods of protein

crystallization, where no gas exchange of the crystallization chamber with the outside environment is desirable (Chayen and Saridakis, Nat. Methods 5, 147-153, 2008). To explore the potential of COPs in protein crystallization, we used a micro fluidics instrument in which the movement and mixing of liquids in COP chips is controlled by centrifugal forces and further described above.

According to the disclosure, the microfluidics structure has the form of a card made of two COP pieces bonded together via a thin COP membrane. One side of the COP card has chambers arranged in rows and horizontal channels. The chambers have dimensions of about 2 x 0.7 x 0.25 mm, corresponding to a volume of about 320 nl. The other side of the COP card contains vertical channels. Connections between chambers and vertical channels and between vertical and horizontal channels are made by a laser that creates openings in the thin membrane separating the two sides of the card. Depending on where the openings are made, specified volumes or metered volumes of liquid can be directed from a chamber in one row to a chamber in the row "below". The movement of liquids is driven by the centrifugal force generated as the cards are spinning in an instrument.

Example II Establishment of microbatch, vapor diffusion and free interface diffusion crystallization protocols

The COP cards according to the disclosure permit the establishment of several protocols for protein crystallization. In the traditional microbatch protocol, protein and precipitant solutions are mixed and the resulting aqueous solution is overlayed by low density paraffin oil, which is impermeable to water vapors (Chayen and Saridakis, 2008). This protocol can be easily established in the COP cards according to the disclosure, simply by directing appropriate volumes of protein and precipitant solutions to the same chamber. Even though openings that are able to direct the liquids in the chambers are never sealed, the very small cross-sectional area of the channels results in very small evaporation

rates; even after months the volume of liquid in the chambers does not change appreciably.

A second protocol established in the COP cards is vapor diffusion (Chayen and Saridakis, 2008). Protein and precipitant solutions are mixed in one chamber, while an adjacent chamber was filled only with precipitant solution. Connections are established between these two chambers by openings above liquid levels.

When the COP cards are incubated at about room temperature, changes in the volumes of the liquids in the two chambers consistent with vapor diffusion are observed within about 6 days. However, at about 4° C, vapor diffusion proceeds more slowly.

A third protocol established in the COP cards is free interface diffusion

(Chayen and Saridakis, 2008). One or more chambers are filled with a protein solution, while an adjacent one or more chamber is filled with precipitant solution.

Again connections via openings between chambers are established, by opening holes below the liquid level. For both the vapor diffusion and free interface diffusion protocols, the rate of diffusion can be controlled by opening more or fewer connections between the chambers (from about 1 to 5 for vapor diffusion and from about 1 to 3 for free interface diffusion).

Example IH

Protein crystallization in COP cards

The suitability of new protein crystallization platforms in the literature is usually documented using proteins that crystallize readily. Following this tradition, a microbatch method is used to monitor crystallization of chicken egg white lysozyme and bovine pancreatic trypsin in the COP cards according to the disclosure. For both proteins crystallization was performed in final volumes of about 200 nl at about room temperature. For lysozyme a grid of final protein concentrations ranging from about 20-60 mg/ml and PME 5000 concentrations ranging from about 4-30%. Crystals formed at protein concentrations between about 22-30 mg/ml and PME concentrations between about 18-30%. For tryspin,

the final protein concentrations ranged from about 15-40 mg/ml and PEG 8000 was used at a concentration of about 30%. Crystals formed at protein concentrations between about 25-30 mg/ml. Lysozyme and trypsin also crystallized in the COP cards according to the invention by the vapor diffusion and free interface diffusion protocols. Because lysozyme and trypsin crystallize readily, other proteins that might be more difficult to crystallize were studied.

In an illustrative embodiment the human p53 tumor suppressor protein was studied. The gene encoding p53 is the most frequently mutated gene in human cancer (Hollstein et al, Science 253, 49-53, 1991). The p53 protein contains a transactivation domain, a sequence-specific DNA binding domain (residues 94- 289) and a homo-tetramerization domain (residues 325-356). The latter two domains are independently-folding domains and their three-dimensional structures have been determined (Cho et al., Science 265, 346-355, 1994; Jeffrey et al., Science 267, 1498-1502, 1995); however, there is no structure of a p53 polypeptide containing both these domains. Polypeptides containing more than one independently-folding domains are generally not easy to crystallize as the linker between these domains imparts conformational flexibility, which inhibits crystallization. In this first illustrative embodiment a p53 polypeptide containing residues 94-291 of human p53 fused to residues 322-356; based on the boundaries of the crystallized DNA binding and tetramerization domains, this polypeptide has a flexible interdomain linker that is 5 amino acids long was studied.

Two amino acid substitutions were introduced in the tetramerization domain of this polypeptide to convert it to a dimerization domain (Davison et al., J.Mol. Biol. 307, 605-617, 2001). In addition, 13 amino acid substitutions were introduced in the DNA binding domain to increase its melting temperature and solubility. The resulting polypeptide retained its sequence-specific DNA binding activity. Studies were undertaken to examine its ability to crystallize in complex with an oligonucleotide containing a p53 DNA binding site using the microbatch and vapor diffusion protocols and three different crystallization buffers. Crystals formed with both protocols after 6 days incubation at about 4° C in the COP cards

according to the invention.

The results show that vapor diffusion yielded p53/DNA crystals with all three crystallization buffers, whereas with the microbatch method p53/DNA crystals were obtained only with two of the three crystallization buffers. The p53 polypeptide/DNA complex also crystallized by the hanging drop vapor diffusion method in 48-well plates under the same crystallization conditions. As a second protein that had not been previously crystallized, studies were conducted on Arabidopsis thaliana Morpheus' Molecule 1 (MOMl), a protein that regulates chromatin structure and gene expression without affecting DNA and histone methylation (Amedeo et al, Nature 405, 203-206, 2000; Habu et al, EMBO Rep. 7, 1279-1284, 2006). An evolutionarily and functionally conserved domain of MOMl maps to a region between about amino acids 1734-1815 (Caikovski et al., PLoS Genet. 4, el 000165, 2008).

Various MOMl fragments in E. coli were expressed and by systematic deletion analysis it was found that a MOMl polypeptide corresponding to residues 1699-1814 of the full length protein is soluble. This polypeptide was purified to homogeneity and examined for crystallization at 4o C by the microbatch, vapor diffusion and free interface diffusion methods in COP cards varying the concentration of the precipitant from 0.2 to 0.4 M. The best results were achieved using the vapor diffusion protocol. This fragment of MOMl also crystallized by the hanging drop vapor diffusion method in 48-well plates under the same crystallization conditions

Example IV Collection of X-ray diffraction data from crystals in COP cards

Crystals that formed in the COP cards could be easily isolated after opening the cards; these crystals could then be cryopreserved, mounted on cryoloops and frozen, thus allowing complete X-ray diffraction datasets to be collected. When there is a need to examine many crystals, the ability to collect X- ray diffraction data while the crystals are still in the COP card could allow

significant savings in time and effort. A robotic arm able to position crystallization multi-well plates in front of an X-ray beam has already been described (Jacquamet et al, Structure 12, 1219-1225, 2004). By comparison to multi-well plates, the geometry of the COP cards used in this study appears well suited for in situ X-ray diffraction analysis.

To examine whether we could actually collect X-ray diffraction data, COP cards containing p53/DNA, MOMl and lysozyme crystals were positioned by the robotic arm in the path of the X-ray beam. The robotic arm was programmed to rotate the card during data collection allowing oscillation of the crystal over a Io range. For all crystals, we could observe diffraction patterns that were of sufficient quality to allow indexing (Figure 3, A and B; and data not shown). The p53/DNA and MOMl crystals exposed to X-rays in situ diffracted to a somewhat lower resolution level than crystals that had been isolated from the cards, cryopreserved, mounted on loops and frozen. For example, cryopreserved p53/DNA crystals diffracted to 3 A, whereas the same crystals in COP cards diffracted to about 4.5 A. We attribute this difference to the temperature shift that occurred during data collection, since the p53/DNA and MOMl crystals formed at 4o C, whereas the in situ data collection was performed at room temperature. In contrast, the lysozyme crystals, which formed at room temperature, diffracted to a resolution of 1.5 A when exposed to X-rays through the COP cards (Figure 8B). To evaluate the quality of data collected from crystals in COP cards, we obtained 45 consecutive X-ray diffraction images, each over an oscillation range of lo, of a lysozyme crystal. The COP absorbed X-rays, but only over a narrow resolution range from 5.4-5.1 A (Figure 8B). At lower and higher resolution ranges, the COP did not compromise data collection, as evidenced both by observing the X-ray diffraction images (Figure 3B) and also by the statistics describing the integration of the X- ray reflection intensities over the 45 frames of collected data (Figure 8C). Data in the resolution range of 40-1.5 A were used to "solve" the structure of lysozyme using molecular replacement; the refined structure had excellent statistics (Figure

8C) and well resolved electron density maps (Figure 8D), especially considering that data from only 45 frames were used for refinement.

The need to optimize the efficiency with which X-ray diffraction quality protein crystals are produced has led to the development of methods for automating the setup of protein crystallization reactions and for reducing the amount of protein required (Chayen and Saridakis, 2008). Most microfluidics systems utilize silicone elastomers as the chip material and have achieved exceptional economies in the amount of protein consumed: in one system 10 nanoliter of protein are required per crystallization condition. However, silicone elastomers are also highly permeable to water vapors and this limits their utility to proteins that crystallize within a few days (Hansen and Quake, Struct. Biol. 13, 538-544, 2003).

Materials that are impermeable to water vapors have also been explored in protein crystallography at a miniaturized scale, but in general these systems require significant human intervention or are compatible with only one method of protein crystallization - usually free-interface diffusion (Ng et al., J. Struct. Biol. 142, 218-231, 2003; Ng et al., Acta Crystallogr. D Biol. Crystallogr. 64, 189-197, 2008). This is because materials that are impermeable to water vapors, such as COPs, are rigid. Unlike, chips made of silicone elastomers, in which liquids can be moved by deforming the chip itself, movement of nanoliter volumes of liquid in rigid chips is not a trivial task. The system we used here solves the "pipetting" problem by opening holes at defined positions to control the volume of liquid to be dispensed and by spinning the cards to move the liquids by centrifugal force. Once the problem of "pipetting" had been addressed, COP-based microfluidic chips could be easily adapted for protein crystallization using several well established protocols, as demonstrated here.

COP cards can help overcome some limitations inherent in microfluidics chips made of silicon elastomers. The first is the issue of water vapor permeability. In COP cards there is very little water evaporation even after months of incubation at room temperature. A second limitation of silicone

elastomer chips is that crystals cannot be readily isolated for X-ray diffraction analysis. This means that new protein crystals have to be obtained using traditional protein crystallization methods. In some cases, it is not straightforward to translate the conditions in which proteins crystallize by free interface diffusion in the microfluidics chip to the conditions in which they will crystallize by the traditional hanging drop vapor diffusion method in multi-well plates. COP cards according to the disclosure overcome this limitation, because the volume of the chambers (320 nl) allows even relatively large protein crystals to form; these crystals can then be easily removed from the COP card to collect X-ray diffraction datasets. Alternatively, limited diffraction data can also be collected from the crystals in situ, because COPs absorb X-rays only within a defined resolution range of about 5.4-5.1 A (Figure *A and Ng et al., 2008).

COP -based microfluidics systems also compare favorably with automated pipetting systems that set up crystallization reactions in multi-well plates (Chayen and Saridakis, 2008). In the latter systems all pipetting steps are performed in an open environment, which allows water to evaporate while the drops are being setup; especially when the volume of these drops is in the nanoliter range. In contrast, in microfluidics systems all pipetting steps are performed in a closed environment, thus eliminating the problem of water evaporation during set-up. The geometry of the COP chambers also facilitates identifying the protein crystals; a task that is much harder with hanging or sitting drops. Based on our experience, we anticipate that COP -based microfluidics will play an important role in protein crystallization efforts.

Example V

A. Protein Sample Preparation

Chicken egg white lysozyme and bovine pancreatic trypsin were purchased as lyophilized powders from Sigma- Al drich (St. Louis, MO. USA) and

AppliChem (Darmstadt, Germany), respectively. Lysozyme (140 mg/ml) was re- suspended in 50 mM NaOAc [pH 4.5]; whereas trypsin (80 mg/ml) was

resuspended in 25 niM Hepes [pH 7.0], 10 niM calcium chloride, 10 mg/ml benzamidine hydrochloride. A polypeptide consisting of residues 94-291 of human p53 fused to residues 322-356 was expressed in E. coli, purified to homogeneity and concentrated to 8 mg/ml in 25 mM bis-tris propane [pH 6.0], 50 mM NaCl, 5 mM DTT buffer. A polypeptide corresponding to amino acids 1699- 1814 of Arabidopsis thaliana Morpheus' Molecule 1 (MOMl) was also expressed in E. coli, purified to homogeneity and concentrated to 6 mg/ml in 25 mM MES [pH 6.0], 200 mM NaCl, 5 mM DTT buffer.

B. Protein Crystallization

Proteins were crystallized either under standard hanging drop vapor diffusion conditions in 48-well plates (Hampton Research, Aliso Viejo, CA, USA) or in COP cards using a dedicated microfluidics instrument (SpinX Technologies, Meyrin, Switzerland). Lysozyme and trypsin were crystallized at room temperature; the MOMl fragment at 4o C; while human p53 was crystallized in the presence of an oligonucleotide containing a high affinity p53 DNA binding site at 4o C. The precipitant solution used for crystallization were as follows: for lysozyme: 4-30% PME 5000, 1 M sodium chloride, 50 mM sodium acetate [pH 4.5]; for trypsin: 30% PEG 8000, 0.2 M ammonium sulfate, 0.1 M sodium cacodylate [pH 6.5]; for p53/DNA complexes: index screen #87 (20% PEG 3350, 0.2 M sodium malonate [pH 7.0]), index screen #89 (15% PEG 3350, 0.1 M succinic acid [pH 7.0]) and index screen #90 (20% PEG 3350, 0.2 M sodium formate [pH 7.0]); for MOMl : 0.2-0.4 M magnesium formate, 0.1 M Tris [pH 8.5]. All crystallization buffers and precipitants were purchased from Hampton Research.

C. Data Collection and Processing

AU data sets were collected at the FIP-BM30A beamline of ESRF

(Grenoble, France; Roth et al, 2002). For in situ data collection COP cards containing lysozyme crystals were positioned in the path of the X-ray beam using

a robotic arm, as previously described (Jacquamet et al, 2004). Reflection data were indexed, integrated and scaled using the program XDS (Kabsch, 1993). The crystals formed in space group P43212 with dimensions a = 79.184 A, b = 79.184 A, c = 38.330 A, and contained one molecule in the asymmetric unit. The coordinates of lysozyme (pdb file laki) were used for molecular replacement using the program AMORE (CCP4, 1994). Then, the structure was refined with the program CNS (Brunger et al., 1998). The electron density maps and the protein atoms were visualized using the program O (Jones et al., 1991).

The principles, preferred embodiments and modes of operation of the presently disclosed have been described in the foregoing specification. The presently disclosed however, is not to be construed as limited to the particular embodiments shown, as these embodiments are regarded as illustrious rather than restrictive. Moreover, variations and changes may be made by those skilled in the art without departing from the spirit and scope of the instant disclosure and disclosed herein and recited in the appended claims.