We intend to develop an improved microfluidic device that allows crystallization experiments to be interrogated directly with X-ray radiation without requiring crystals to be removed from the well. This will allow experimenters to assess the outcome of crystallization experiments on the basis of diffraction data rather than arbitrary optical judgments. Identification of conditions for growth of X-ray diffraction quality crystals continues to be a limitation in macromolecular crystallography. This is a particular problem in the case of samples that can only be prepared in limited quantities, such as membrane proteins and multi-component macromolecular complexes. Fluidigm Corp. has developed and commercialized the TOPAZ(r) system, that uses microfluidic chips for crystallization using minimal (<10nl) sample volumes per experiment. The new chip product we will develop will be fully compatible with the TOPAZ crystallization system. The chip will be designed to be manufactured in large quantities, and with a cost that is reasonable for the end user. During Phase I, we designed microfluidic chips from which diffraction data could be collected at both ambient and cryogenic temperatures. Phase I experiments demonstrated the ability to collect diffraction data from crystallization experiments in situ, allowing rapid determination of 1) whether crystals are protein or salt, and 2) the diffraction quality of the crystal. During phase II, in addition to development of the chip, we intend to modify the end stations of Beamlines 8.3.1 and 12.3.1 at the Advanced Light Source (ALS) at Lawrence Berkeley National Laboratory (LBNL). The goniometers will be modified to hold the TOPAZ carrier and position wells for interrogation by the X-ray beam. Appropriate software will also be developed to automate control of the positioning and alignment of the chip at the beamline. The TOPAZ goniometer will be designed to be compatible with other X-ray diffractometers. We have shown that TOPAZ chips containing crystals can be transported at room temperature with little apparent damage to the crystals. We will characterize this transport behavior with samples from collaborator labs, and investigate the development of a diffraction-based chip screening service at LBNL. The combination of technical and scientific strengths at Fluidigm and LBNL will enable development of a robust, commercially-available product suitable for diffraction-based screening of macromolecular crystals. This will provide not only a significant enhancement to currently available crystallographic methods, but also an increase in the rate of successful structure determination. 7. Project Narrative: Determination of protein and nucleic acid structures has had a profound influence on understanding and developing treaments for human disease. This project will develop a commercial product that will eliminate a significant bottleneck in determination of these structures-assessing the quality of crystals used for diffraction studies immediately after the conditions to grow them have been identified. Moreover, this will be carried out in a microfluidic chip, using tiny volumes of sample, and without requiring the crystals ever to be touched or handled before data collection. ? ? ?
| Schoeffler, Allyn J; May, Andrew P; Berger, James M (2010) A domain insertion in Escherichia coli GyrB adopts a novel fold that plays a critical role in gyrase function. Nucleic Acids Res 38:7830-44 |
