SBIR-STTR Award

?Crystallization, followed by subsequent structure determination, is a major step in understanding the structure-function relationship of macromolecules. Understanding macromolecule structure has become a key part in the development of new pharmaceuticals, and is a major area of NIH research. Crystallization however is also the rate limiting step, despite technological efforts to automate the set-up and crystallization data acquisition processes. Macromolecule crystallization conditions are arrived at by screening experiments, where the target material is subjected to typically hundreds to even thousands of different chemical cocktails. In most cases screening experiments fail as they do not result in a crystal. We propose that the experiments contain useful information about the targets behavior in response to the imposed conditions, and that the results can be analyzed to extract the relevant parameters for guiding subsequent crystallization trials. No screen or group of screens can systematically cover the chemical space for protein crystallization, and we hypothesize that in the absence of clear positive hits scored results can be analyzed to determine these factors. As a test of this a preliminary screening results analysis program was written and tested using three hyperthermophile proteins, one each a very easy, moderate, and a very difficult crystallizer. Characterizations were on the basis of each proteins behavior in a single crystallization screen. The analysis is called the Associated Experimental Design (AED) approach. The analysis identified the most significant factors and a 96 condition screen based on those factors was prepared for each protein and set up. Crystals were obtained for all three proteins, and none of the crystallization conditions duplicated those in the original screen. Our Phase I goal is to further improve the preliminary AED software. The Phase I improvements are to include up to three or four screens in the analysis function, to treat salt anions and cations separately in the analysis, to output a 96 condition screen for each protein composed of the found significant factors for that protein, and to test a revised scoring scheme. For simplicity th same screens will be used throughout the Phase I effort. A pool of test proteins will be employed, 1/3 each being classified as easy, moderately difficult, and difficult crystallizers based upon their performance in screening trials. Each iteration of the software will be used with the test proteins original scoring data and optimization screens set up. The optimization results will be fed back to further guide the AED software development. Proteins classified as difficult are anticipated to require two or more optimization rounds. Based on the preliminary results the AED method shows considerable promise. A major advantage of this approach is that it will fit into existing practice, making use of the data already generated in crystallization screening. Success with this approach will increase the number of hits generated and greatly reduce the time and effort required for macromolecule crystallization.

Public Health Relevance Statement:


Public Health Relevance:
Successful crystallization and X-ray data analysis provides important three-dimensional information on the macromolecules structure-function relationship, important to the design of pharmaceuticals. Screening experiments to identify crystallization conditions typically return non crystalline results. This proposal is to develop software for the analysis of screening data scores to identify likely significant crystallization factors, providingan analytical basis for subsequent experiments, and thereby increasing the chances of success.

Project Terms:
Anions; Area; Automation; Back; base; Behavior; Cations; Chemicals; Computer Programs and Programming; Computer software; cost; Crystallization; Data; data acquisition; Data Analyses; Data Set; design; Development; Experimental Designs; Failure (biologic function); Feedback; feeding; follow-up; Goals; Housing; hyperthermophile; improved; Lead; macromolecule; Methods; Modeling; Numerical value; Outcome; Output; Performance; Pharmacologic Substance; Phase; Probability; Process; programs; protein structure; Proteins; public health relevance; Research; research study; response; Roentgen Rays; Salts; Scheme; screening; Screening Result; Series; Set protein; Sodium Chloride; software development; Solutions; Structure; Structure-Activity Relationship; success; Test Result; Testing; Time; tool; Training; United States National Institutes of Health; Work; Writing; X-Ray Crystallography

Crystallization, followed by subsequent structure determination, is a major step in understanding the structure-function relationship of macromolecules. Understanding macromolecule structure has become a key part in the development of new pharmaceuticals, and is a major area of NIH research. Crystallization however is also the rate limiting step, despite technological efforts to automate the set-up and crystallization data acquisition processes. Macromolecule crystallization conditions are arrived at by screening experiments, where the target material is typically subjected to hundreds or even thousands of different chemical cocktails. In most cases screening experiments fail as they do not result in a crystal. We propose that screening experiments contain useful information about the target proteins behavior in response to the tested solution conditions. No screen or group of screens can systematically cover the combinatorial chemical space for protein crystallization, and we hypothesize that in the absence of clear positive hits scored results can be analyzed to determine these factors. The analysis method developed is called the Associated Experimental Design (AED) approach. The analysis identified the most significant factors and a 96 condition screen based on those factors is prepared for each protein and set up. In the (ongoing) Phase I effort the AED software is being progressively evolved, adding functions for aiding in prioritizing the screen factors employed for likely success in crystallization. The software is written to not duplicate input conditions for a given protein in the output; i.e., all output conditions are new combinations of high probability factors as determined from the analysis. The software has been tested with 23 proteins to date. Of the 5 proteins that did not give crystals upon initial screening, 2 gave crystals from screens developed on the basis of the AED analysis. Of the 18 remaining proteins, 72% gave as many or more crystals in the single AED based screen than were obtained in the 4 x 96 condition screens. One of these 18 proteins was the RrP41-RrP42 archaeal exosome catalytic core complex. Based on the Phase I results the AED method shows considerable promise. A major advantage of this approach is that it fits into existing practice, making use of existing materials, methods, and data routinely generated in crystallization screening. The AED software can be used with any imaging system that gives a scored assessment of the results for each trial, including manual scoring by a user with a simple low power microscope. The Phase I results also showed that it can be used with a reduced, more granular, scoring scale. Success with this approach will increase the number of hits generated and greatly reduce the time and effort required for macromolecule crystallization. The proposed Phase II effort is to build upon the successful approach developed in Phase I and further develop the analytical methods employed.

Public Health Relevance Statement:
Successful crystallization and X-ray data analysis provides important three-dimensional information on the macromolecules structure-function relationship, important to the design of pharmaceuticals. Screening experiments to identify crystallization conditions typically return non crystalline results. This proposal is to further develop and test software for the analysis of screening data scores to identify likely significant crystallization factors, providing an analytical basis for subsequent experiments, and thereby increasing the chances of success.

Project Terms:
analytical method; Area; Automation; base; Behavior; Businesses; Catalytic Domain; Chemicals; combinatorial; Complex; computer program; Computer software; Contracts; cost; Crystallization; Data; data acquisition; Data Analyses; Data Set; design; Development; Encapsulated; Excision; exosome; Experimental Designs; Failure; file format; Formulation; Goals; Guidelines; Housing; hyperthermophile; imaging system; improved; Laboratories; Licensing; Link; literature survey; macromolecule; Manuals; Manufacturer Name; meetings; Methods; microbial; Microscope; Outcome; Output; pathogen; Pharmacologic Substance; Phase; Probability; Process; programs; protein structure; Proteins; proton-translocating pyrophosphatase; Research; research study; response; Roentgen Rays; screening; Screening Result; Series; Services; Set protein; software development; Source; structural biology; Structure; Structure-Activity Relationship; success; Testing; Thermococcus; Time; tool; United States National Institutes of Health; Vendor; Work; Writing; X-Ray Crystallography