Abstract:
A biological crystal formation screening apparatus uses an x-ray diffraction technique to analyze the sample containers of a sample tray for the presence of crystal formation. An x-ray source is directed toward a sample under investigation, and a two-dimensional x-ray detector is located to receive any diffracted x-ray energy. A positioning apparatus allows the different sample containers of a tray to be sequentially aligned with the source and detector, allowing each to be examined. Various techniques for interpreting the detector output data are also provided.

Description:
FIELD OF THE INVENTION  
         [0001]    The present invention relates generally to the field of structural genomics and, more specifically, to the use of crystallography to examine protein crystals for genomic research.  
         BACKGROUND OF THE INVENTION  
         [0002]    In biological research, particularly in the field of genomics, crystallography is a tool used to examine the characteristics of proteins. However, such proteins are typically developed in a liquid medium, and therefore must be crystallized in an orderly fashion before detailed crystallography techniques may be used. A typical method for crystallizing such proteins is through vapor diffusion. In a method known as the “hanging drop” method, a well solution is placed in the many separate sample wells of the sample tray. For each sample well, a drop of protein liquid is applied to a slide, which is placed over the sample well with the drop hanging down toward the well solution. Because of different relative concentrations of the well solution and the droplet solution, over time, liquid diffuses out of the droplet and into the sample well, resulting in the crystallization of the protein on the surface of the slide.  
           [0003]    Depending on the conditions under which the crystallization process takes place, the formation of a crystal may take anywhere from hours to months. While some crystals are visible to the naked eye, the sample slides must usually be examined with a microscope one at a time to determine whether protein crystallization has taken place. Of course, for those protein samples that have not yet crystallized, the slides must be reexamined on a regular basis until the crystallization is observed. For a relatively large number of samples, this is obviously a long and labor-intensive process.  
         SUMMARY OF THE INVENTION  
         [0004]    In accordance with the present invention, a screening apparatus is provided for monitoring crystal formation in a crystal growth medium that makes use of an x-ray source and detector. X-ray energy from the x-ray source is incident on a sample container and undergoes diffraction if in the presence of a crystal structure. Any such diffracted x-ray energy is detected by the x-ray detector, the output of which is indicative of the presence or absence of such a crystal structure. In this way, one may determine whether any significant crystal formation has taken place in the crystal growth medium, without the need for visual examination of the sample container. This is particularly useful for the examination of biological crystal formation common in genomics research.  
           [0005]    In a preferred embodiment, the screening apparatus also includes a positioning apparatus for locating the sample container relative to the x-ray source and x-ray detector. The positioning apparatus has a support that is remotely movable in at least two dimensions, allowing the precise positioning of the sample container relative to the x-ray source and detector. This is particularly useful in the preferred embodiment of the invention, in which the sample container is one of a plurality of sample containers each having a separate crystal growing medium. The sample containers may be part of a contiguous array, such as in a sample tray having an array of sample wells. In such a case, the positioning apparatus may be used to move the sample containers so as to position them sequentially relative to the x-ray source and detector, thereby allowing sequential examination of the sample containers. In addition, the source and detector may be arranged to operate in reflective mode or in transmission mode. If used in transmission mode, the positioning apparatus preferably has an open section located between the source and a sample well under investigation so as to not interfere with the source x-ray energy.  
           [0006]    The x-ray source and detector may be arranged such that the exposure of x-rays from the source covers a two-dimensional area of the sample container being examined, in particular, an area over which any significant crystal formation would be expected to appear. The detector, similarly, is a two-dimensional detector, providing simultaneous detection of x-ray energy diffracted from a similar two-dimensional region of the sample container. Therefore, a simultaneous set of pixel intensities may be collected that is indicative of any presence of crystal structures across the two-dimensional area of the sample container under investigation.  
           [0007]    A control apparatus is preferably used to control the various aspects of the screening apparatus, including the triggering of the x-ray source and the collection and processing of data from the detector. The control apparatus may also be used to control the positioning apparatus to synchronize the alignment of the various sample containers in an array with the operation of the x-ray source and detector. In this way, a the system may be used to automatically analyze the entire array of sample containers to determine which, if any, show the formation of any significant crystal structure.  
           [0008]    In addition to the structural aspects of the invention, various techniques are also provided that may be used to evaluate the intensity data from the pixels of the detector to make a determination of whether or not a crystal is present. One such technique involves determining the number of pixels having an intensity level exceeding a minimum pixel intensity level and comparing that number to a predetermined minimum number selected as being indicative of the presence of said crystal structure. In another method, the outputs from a predetermined number of pixels having the highest intensity levels are averaged and compared to an overall average intensity value of all the pixels. In yet another method, the pixel intensity values that are indicative of the presence of a crystal peak in the detected spectrum are isolated and integrated. This integrated crystal peak intensity is then compared to an integrated intensity of all the detector pixels. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0009]    The above and further advantages of the invention may be better understood by referring to the following description in conjunction with the accompanying drawings in which:  
         [0010]    [0010]FIG. 1 is a schematic side view of a screening apparatus according to the present invention;  
         [0011]    [0011]FIG. 2 is a graphical view of a two-dimensional set of x-ray intensities resulting from x-ray scattering from an amorphous surface;  
         [0012]    [0012]FIG. 3 is a graphical view of a two-dimensional set of x-ray intensities resulting from diffraction from a crystal structure and background scattering from amorphous materials;  
         [0013]    [0013]FIG. 4 is a schematic top view of a sample tray having an array of sample wells;  
         [0014]    [0014]FIG. 5 is a graphical view of an absolute pixel intensity method of determining the presence of a crystal structure from a two-dimensional set of intensity data produced with a screening apparatus according to the present invention;  
         [0015]    [0015]FIG. 6 is a graphical view of an relative pixel intensity method of determining the presence of a crystal structure from a two-dimensional set of intensity data produced with a screening apparatus according to the present invention; and  
         [0016]    [0016]FIG. 7 is a schematic view of an alternative embodiment of the invention in which transmission mode x-ray diffraction is used with a sitting drop sample well arrangement. 
     
    
     DETAILED DESCRIPTION  
       [0017]    Shown in FIG. 1 is an x-ray screening apparatus that may be used to identify the crystallization of protein samples in a sample tray  10 . In the figure, the sample tray  10  is shown in a cross-sectional side view, so that the contents of one row of sample wells  12  are apparent. Contained within each of the wells  12  is a well solution  14  that induces vapor diffusion from a sample drop located in the underside of a slide (or mylar film)  16  covering the top of the well. The process of vapor diffusion is well known in the art, and will not be repeated in any significant detail herein. However, in the present embodiment, rather than use visual inspection to determine when crystallization has occurred, the samples are examined using a diffraction-based technique.  
         [0018]    In the embodiment of FIG. 1, the sample tray is mounted on a translation table  18  that is adjustable in three dimensions. The translation table allows the sample tray to be repositioned within a three dimensional area in order to align and realign the sample wells as desired. Control of the movement of the translation table  18  is preferably automated, and responsive to a control program for examining the samples. Movement of the translation table  18 , and thereby the sample tray  10 , allows it to be repositioned relative to x-ray source  20  and two-dimensional x-ray detector  22 .  
         [0019]    In the preferred embodiment, x-ray source  20  is a sealed tube or a rotating target generator that produces x-ray radiation in a wavelength range of approximately 0.5 to 2.3 angstrom. The source  20  also includes appropriate x-ray optics to condition the x-ray beam into a specified beam size, spectrum and beam profile. The detector  22  is any of a number of known two-dimensional x-ray detectors that can simultaneously detect the intensity of x-ray energy with a number of pixels across a two-dimensional area. In operation, each sample well is scanned one at a time. The scanning operation is controlled by a system controller  19  that controls the firing of the x-ray source  20 , the data collection from detector  22  and movement of translation table  18 . Preferably, the controller runs an automatic scanning routine that provides sequential scanning of all (or selected) sample wells, and corresponding data collection and processing. Control apparatus such as these are known in other fields, and are easily adaptable to the present invention by one skilled in the art. In operation, the translation table  18  moves the tray so that a sample well  12  to be examined is in the path of an x-ray beam from the source  20 . The incident x-rays pass through the coverslips or mylar film and are incident upon the hanging droplet within the sample well. The cross-sectional area of the incident beam is large enough that a single exposure will reach any part of the well in which a crystal might form. If a crystal is present, it diffracts the x-rays from source  20  in the direction of the detector  22 , where x-ray intensities are detected across the detector surface.  
         [0020]    As each sample well is scanned, a data frame is collected for it that is representative of two-dimensional distribution of x-ray intensities across the detector surface. Based on the content of this data frame, a determination may be made regarding the degree to which any crystal structure has formed in the sample well under investigation. Shown in FIG. 2 is a graphical depiction of the pixel intensity distribution in a data frame for which there is no significant crystal formation. Materials surrounding the sample material, such as the crystallization plate, coverslips or mylar film and the liquid are amorphous, so that the x-rays scattered by them are randomly distributed. This results in a spectrum as shown in FIG. 2, in which there is a relatively consistent distribution of x-ray energy across the two-dimensional space.  
         [0021]    When there is a significant degree of crystallization in a sample well, the crystal will diffract x-rays toward the detector  22 . The diffracted x-rays form sharp intensity peaks much more intense than the background caused by scattering from amorphous materials. The particular crystallinity condition within each screening spot can be determined by the number and intensity of the peaks. An example of such a spectrum is depicted graphically in FIG. 3. As shown, within the background noise caused by the scattering from amorphous materials are several distinct diffraction peaks. The presence of these peaks may be used as part of an automated analysis program for screening the protein samples.  
         [0022]    Shown in FIG. 4 is a schematic top view of a sample tray having a 6×9 array of sample wells. Those skilled in the art will recognize that this particular number of sample wells is for illustrative purposes only, and the actual sample tray may have any number of sample wells, and will likely have many more than are shown. From this figure, it may be understood that the translation table  18  shown in FIG. 1 may be used to move the sample so as to sequentially align the sample wells with the x-rays from source  20 . The instrument center is defined by the crossing point of the incident x-ray beam and a center line of the detector. The system automatically and sequentially moves the tray so that the location of each droplet is sequentially moved to the instrument center. As each of the sample wells is aligned with the source, a data set is collected with the detector  22 , and stored for analysis purposes. Using an arrangement as shown in FIG. 4, the progress in the movement of the tray may be broken down by a series of steps in two dimensions. Once the tray is located relative to a starting location, such as point  24 , oriented at the instrument center, subsequent movements of the tray may be a series of predetermined steps, such as an x-dimension step  26 , or a y-dimension step  28 . With each step, a scan is performed of the sample well located at the new location, and the movement continues until an end location, such as location  30 , is reached. At this point data collection is complete. Of course, those skilled in the art will recognize that any desired scanning pattern may be used as necessary, and the provision of a user interface that allows custom table movement is fully anticipated.  
         [0023]    Once the desired droplet scan data is collected, it must be analyzed to determine a degree of crystallization in each of the sample wells being examined. The scanning portion of the invention may be used with any desired data analysis techniques. However, several possible techniques are disclosed herein.  
         [0024]    A first method of crystal peak identification may be referred to as the “absolute pixel intensity” method. The two-dimensional detector  22  has a given number of detection pixels, each of which detects a particular x-ray intensity each time a sample well is scanned. If pixel intensity is identified by a finite number of intensity levels, called “pixel counts,” than a data set may be collected that correlates each pixel with a corresponding pixel count. A determination of crystal presence may then be based on meeting a threshold number of pixels having a minimum intensity level. That is, the presence of a crystal will be assumed if at least a minimum number of pixels n have at least a minimum pixel count c. A graphical interpretation of this method is depicted in FIG. 5. In this figure, the horizontal axis represents pixel count while the vertical axis represents a number of pixels for a corresponding pixel count. The dashed line in the figure depicts the outcome if no crystal peaks are detected. As shown, none of the pixels register the minimum pixel count c, and a determination is therefore made that no significant crystallization has occurred at this droplet site. The solid line in the figure depicts the outcome when a sufficient number of crystal peaks are detected. As shown, the resulting curve includes more than n pixels with a minimum pixel count of c, and so a determination is made that sufficient crystallization has occurred at this site.  
         [0025]    Another method of identifying crystal formation may be referred to as the “Relative Pixel Intensity” method. It relies on measuring the intensity of the brightest pixels relative to the average pixel intensity. In this embodiment, a predetermined number n of pixels are selected for having the highest intensity, and the average intensity I n  of these n pixels is compared to the average intensity I o  of all the pixels. If the ratio of the intensity of the high intensity pixels to the average pixel intensity is at least a predetermined value k, than sufficient crystallization is deemed to have occurred. The corresponding conditions may therefore be represented as follows: 
         If I n /I o ≧k, crystal is found 
         If I n /I o &lt;k, no crystal is found 
         [0026]    The graphical representation of FIG. 6 shows the relative difference between the intensity averages I n  and I o  in a depiction of the pixel intensities arranged from highest to lowest along the horizontal axis.  
         [0027]    Yet another method of determining the presence of crystallization may be referred to as “integrated peak intensity.” This method recognizes that, when crystallization is present, there is a wide intensity difference between the sharp peaks resulting from the crystal diffraction, and the background intensity due to amorphous scattering. Certain known mathematical models are available by which the pixel data from the diffraction peaks may be separated from the pixel data from the background. Once separated, the integrated intensities for all of the crystal peaks may be compared to the total integrated intensity in the data frame. If a ratio of the integrated intensity (I c ) of the crystal peaks to the integrated intensity of the entire data frame (I t ) exceeds a predetermined value p, then sufficient crystallization is deemed to have occurred. This relationship may therefore be represented as follows: 
         If I c /I t ≧p, crystal is found 
         If I c /I t &lt;p, no crystal is found 
         [0028]    Those skilled in the art will recognize that many different criteria may be used to determine the presence of sufficient crystallization once the data from the detector pixels is collected. The particular method of determination may be customized to the systems and experiments of particular users.  
         [0029]    While the embodiment of FIG. 1 demonstrates the use of the screening technique of the present invention using a system in “reflection mode,” it is also possible to use a “transmission mode” arrangement. Such an arrangement is shown schematically in FIG. 7. Also demonstrated in this figure is the use of the present invention with the “sitting drop” type of vapor diffusion. Whereas the “hanging drop” method has the sample solution droplet positioned on the underside of a slide or other covering over the sample well, the “sitting drop” method locates the droplet on a separate platform elevated above the well solution  114 . However, it should be noted that the present invention may be used in either reflection mode or transmission mode with either of the hanging drop or sitting drop arrangements.  
         [0030]    In the embodiment of FIG. 7, an x-ray source  120  is located to the opposite side of the sample tray from a detector  122 . At least the relevant portions of the sample tray are amorphous and effectively transparent to x-ray energy so that the x-ray energy from source  120  interacts with the protein sample in the well under investigation. The translation table  118  shown in the embodiment of FIG. 7 has a cutaway portion beneath the sample wells, and the sample tray is supported along its edges. This avoids the obstruction of the source  120  by the translation table. However, those skilled in the art will recognize that a different translation table could be used as long as only x-ray transparent material separated the source  120  and the wells  112 .  
         [0031]    When there is a significant degree of crystallization in a sample well  112 , the crystal will diffract x-rays toward the detector  122 . The diffracted x-rays form sharp intensity peaks much more intense than the background caused by scattering from amorphous materials. This diffraction spectrum is similar to that developed when using the invention in reflection mode, but the relative diffraction angles for the wavelengths being detected are obviously different in the two arrangements. In each case, the detected wavelength peaks will depend on the relative orientation of the components, the material under investigation and the x-ray wavelengths from the source  120 . As in the embodiment of FIG. 1, it is preferred that the functions of the system, including operation of the x-ray source, movement of the translation table, and collection and processing of data from the detector  122  are coordinated by a system controller  119 . Naturally, other uses of the present invention that vary from the embodiments shown are anticipated.  
         [0032]    While the invention has been shown and described with reference to a preferred embodiment thereof, those skilled in the art will recognize that various changes in form and detail may be made herein without departing from the spirit and scope of the invention as defined by the appended claims.