ProCreator: Automating the Creation of Buffer Matrices for Protein Crystallization

Overview

The process of creating buffer matrices for protein crystallization experiments has proven to be a tedious task for structural biologists. Therefore, many have turned to commercial buffer kits for a quick solution to this problem. Commercial kits, however, are static and prevent scientists from manipulating the conditions that will be presented to a particular protein. Because custom “kits” are often desired, a software application, ProCreator, has been developed at Merck & Co, Inc. to automate the process with the aide of a Tecan Genesis instrument. Users can design a two-dimensional matrix of buffer conditions. This matrix may contain any number of liquids necessary to achieve a custom and ideal buffer kit. In addition, key parameters, such as concentrations and/or pH, can be varied across and/or down a plate. ProCreator's calculations have replaced the tedious process of dilutions and pH measurements to allow easy preparation of diverse conditions. These values for buffer conditions were found to be < 0.2 pH units from the theoretical value. Through minimal user interaction and a graphical interface, users can design and create a protocol within minutes to fit their needs. As a result, automating the creation of custom buffer conditions will allow for more specific screening of proteins and an expected higher yield of crystals in years to come.

Introduction

The problem facing structural biologists proves to be a complex task to undertake. Protein crystallography has traditionally been a time-consuming, very precise science. The cornerstone of this scientific procedure is vapor diffusion of a “mother-liquor” with the protein being crystallized. This “mother-liquor” consists of a buffer and desired reagents that theoretically induce crystallization. Because the mechanisms for crystallizing most proteins is relatively unknown, structural biologists have taken a trial-and-error approach to finding ideal conditions for crystallizing a particular protein. This trial-and-error approach is quite tedious, time-consuming, and costly. In conjunction with the strides made in liquid handling over the past few years, protein crystallographers began to look for ways to automate the process and reduce the time needed to find a “hit”, in this case, a crystal. One possible solution is to use commercial buffer kits that provide commonly used buffer conditions to trigger crystallization. The setback with this approach is that commercial kits are static, thereby leaving scientists with little manipulation on the buffer condition that is presented to the protein. Therefore, another solution is required to achieve flexibility in experimentation. A two-dimensional matrix of buffer conditions in a plate format is a promising alternative.

The creation of this two-dimensional matrix of buffer conditions requires automation via a liquid handler, a Tecan Genesis. The base software for the instrumentation, Gemini, produces another challenge; the software was designed for “plate-based” automation, however, this process required “sample-based” automation. In other words, because every well of every plate is essentially different, every tip must be able to work independently in order not to sacrifice efficiency. The solution of this problem is found in Gemini's worklist capabilities. A worklist is an external file that is read within a base Gemini script that sends the appropriate commands to the instrument. With the use of this worklist, one can control each tip individually to create the desired matrix.

However, because the matrices can be quite complex, the creation of the worklist file is not easily created by users. Therefore, a custom application is needed to generate this file in the desired format. Merck & Co., Inc., developed a software application, ProCreator, for the creation of mother-liquor protocols. ProCreator is designed to incorporate Gemini's worklist functionality with extensive flexibility in the design of protocols. In addition, it enables users to generate reusable protocols without the reliance on a network, database, or scheduling software.

Methodology

A Tecan Genesis Robotic Sample Processor (RSP 100) was used as the vehicle for liquid-handling. It allows for individual control of each tip on the 8-tip head. This functionality was vital for the creation of custom, “homemade” buffer kits. Gemini, Tecan's Genesis software, was selected as the base software for the instrumentation.

Using Gemini's worklist functionality, it was possible to control individual tips and manipulate their motions for this custom layout. Therefore, a simple solution for generating work-lists was devised. A dynamic-link library (DLL) was designed in Visual Basic 6 (VB) to generate files in the format of Gemini worklist. This DLL could be integrated into any VB code to allow similar functionality in other applications.

In addition to the basic worklist format, complex functionality required by crystallographers had to be implemented into ProCreator. These requests are summarized in Table 1.

The graphical user interface was given an Outlook-style to allow users greater familiarity when navigating through the application. In addition, the software was designed to be independent of databases or scheduling software. Gemini is the only program required to be installed on the client computer alongside of ProCreator. In order for ProCreator to populate itself with pertinent information about Gemini racks and carriers, ProCreator requires a version of Gemini installed on the computer. ProCreator works reliably with Version 3.4–4.0 of Gemini.

Simple and complex mathematical calculations were a large part of the functionality of ProCreator. In order to provide the scientific aspect of the software, fundamental chemistry equations were used. For the variation of concentrations, simple dilution and molarity equations were used. In the case of variations of pH, more complex systems of equations, incorporating the Henderson-Hasselbach equation (Equation 1), were solved.

Tip optimization also incorporated complex mathematical algorithms. It was necessary to design the logic behind the tip optimization so that it could be applied to all Gemini rack configurations possible. The optimization would reduce the number of moves and wash steps, without sacrificing the reliability or introducing the possibility of cross-contamination.

User Interface

The graphical user interface was given an Outlook-style in order to give users greater familiarity when navigating through the application. ProCreator consists of two basic screens, illustrated in Figure 1 and Figure 2. Figure 1 illustrates the Destination Parameters. The user must designate a “Label” for the destination that corresponds to RackLabel in Gemini. Also, the destination rack type, the number of liquids in the protocol and the total volume per well must be specified.

The second major screen is for reagent parameters. One of these forms will be required for each liquid specified in the protocol. A reagent can serve four purposes. It can be added at a constant volume to each well; it can vary in concentration across or down a rack; it can vary in pH across or down a rack; and if it is the final liquid defined in a protocol, it can be used to fill the wells up to the total volume desired. Based on the function of the reagent, certain parameters exclusive to the function are required. These parameters are summarized in Table 2.

An illustration of the Reagent Parameter screen is shown in Figure 2. If you notice, the number of rows and columns to pipette are specified by the user, as well as the starting row and column for both the source and destination. The highlighted wells represent chambers that will be filled with the current liquid. This graphical representation allows users a better understanding of the protocol that is being created.

Testing

ProCreator was tested to ensure that all commands and calculations were correct. The software was used to generate > 50 worklists, incorporating most possibilities required by the users. Each worklist was hand-checked to ensure accuracy and precision of the generation step. The application worked without failure. In addition, the worklists were loaded on a Tecan Genesis and executed to test the motions. The tests were completed without failure. Finally, the application's calculations were tested by using a pH probe and testing theoretical values with those pipetted manually with the values generated by ProCreator and by the Tecan Genesis using the generated worklist. The results of that test are illustrated on Figure 3. The results yielded an accuracy to within 0.2 pH units. This slight variation from the theoretical values could have easily been caused by CV's in the pipetting apparatus.

Conclusion

Procreator allows for maximum flexibility and ease-of-use for protein crystallographers. Through minimal user interaction and a graphical interface, users can design and create a protocol within minutes to fit their needs. As a result, automating the creation of custom buffer conditions will allow for more specific screening of proteins at a higher rate thereby yielding a greater number of expected crystals. Based on the results from the extensive robotic testing and the buffer tests that yielded pH measurements < 0.2 pH units from theoretical expectations, ProCreator is shown to be a robust and reliable solution for creating protocols and matrices for protein crystallography or any other application requiring “sample-based” automation.