Enabling membrane protein structural analysis: Tools for capillary diffusion crystallization and remote in situ diffraction experiments

Project SummaryThis proposal is to develop an integrated capillary counter diffusion-based approach to in situ collection of macromolecule diffraction data. The method to be developed will enable both cryogenic and ambient temperature data collection, with the latter being critical to structure-based drug design. The approach is based on a capillary that is pre-mounted in a holder that facilitates its being moved from loading to crystal growth to diffraction analysis without ever having to physically touch or directly manipulate the crystal. The capillary is closed at one end and is filled by centrifugation, which method enables filling with very high viscosity solutions. Crystallization is by capillary counter diffusion (CCD), a method that often results in the growth of crystals, often multiple crystals, that fill the internal diameter of the capillary.The immediate research goals of this effort are the development of and improvements to the mounted capillary design. These are more specifically focused on the capillary holder design, how the capillary is mated to the holder, the lower size limits for the capillary internal diameter that can be employed, the capillary materials that can be used, and the methodology by which the capillary is secured to the holder. Different solution holder designs, by which the mounted capillary is introduced to the precipitant solution for crystallization, will be investigated, as well as the holder used for centrifugally filling the capillary. As the designs are implemented they will then be tested by the growth and subsequent diffraction of soluble and integral membrane protein crystals.This proposal is a collaborative effort between the PI, Mr. Richard Howells of Crystal Positioning Systems, Dr. Marc Pusey of the University of Alabama in Huntsville, and Dr. Aina Cohen of Stanford Synchrotron Radiation Laboratory. RH will be responsible for all machine shop work, fabricating parts to designs as suggested by MP and AC. MP will be responsible for assembly of the mounted capillaries, preparation of the proteins to be used as test materials, setting up the crystallization experiments, and all functional testing of the hardware developed. AC will be responsible for all beamline-specific aspects of this effort; to test the mounted capillaries on the beamline and to ensure their compatibility with sample exchange robots employed at synchrotron sources. Diffraction data collection and data analysis will use the SSRL beamlines and computing infrastructure. Data and structure analysis will be overseen by RH with input from MP and AC. AC and AH, together with MP, will provide input to RH for improvements to the designs, and will then test those improvements as they are fabricated.Project NarrativeThis research directly addresses the ability to carry out structure-based drug design targeting macromolecules, through development of a means to grow protein crystals and carry them through to X-ray diffraction analysis without ever having to physically touch them. The methods to be developed are proposed to be applicable to both soluble and integral membrane proteins, with diffraction data being collectable either in situ and remotely at either room or cryogenic temperatures and with full rotational access.