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Professor Rob Thorne, Cornell Physics Dept., is actively developing tools and techniques for protein crystallography, as well as studying the physical processes occurring in crystals exposed to X-rays and/or subjected to changes in temperature. A recent area of focus is the growth of protein crystals. A common, well-developed, technique for this purpose involves the diffusion of solvent vapor, usually water, between a drop of solution containing protein and a "reservoir" solution of different composition, e. g. higher in a precipitant concentration. The plates commonly used for vapor diffusion have some deficiencies, particularly when used in a high-throughput operation where it is desired to evaluate crystals in situ rather than putting effort into extracting putative crystals, many of which will turn out to be useless. A recent publication from the Thorne group addresses this problem.

Development of High-performance X-ray Transparent Crystallization Plates for in situ Protein Crystal Screening and Analysis. A.S.M. Soliman, M. Warkentin, B. Apker, and R.E. Thorne, Acta Cryst. (2011)D67, 646-656.

The typical crystallization plates are made of relatively thick plastic, which produces a high background if the plate is put in an X-ray beam to check whether a crystal diffracts. Moreover, the drops of protein and reservoir solution are free to move around if the plate is turned from horizontal to vertical (to pass a horizontal X-ray beam through it), meaning that they may mix, causing crystals to be disturbed, perhaps dissolved. These problems are solved with the new TDP plate, which makes use of a very thin film of COC (cyclic olefin coplymer) plastic, patterned with ridges which act to pin drops in place, even when the film is rotated to a vertical position, or even upside down (producing hanging drops, which are commonly used with manual mounting but seldom with automated systems). The film is mounted on the bottom of an open mesh frame, for stability, and the top of the frame is sealed with another thin film to prevent evaporation. The new design has the potential to make in situ, low background, examination of possible crystals a routine part of high-throughput protein structure initiatives.


Prototype TDP plate. a) injection-molded frame; b) 3 and 30 uL water drops on microembossed film; c) diagram of ring pattern in each 2-drop cell; d) auto-dispensed drops on prototype plate: 0.2 and 2 uL protein drops (red)) and 15 uL reservoir (blue) drops; e) same drops after inverting the plate; f) lysozyme crystals grown in the plate.

Thorne has also put a great deal of effort into developing tools to work with the crystals once they are grown, and has created a company, MiTeGen, to sell these to the public and to continue improving the devices. Beginning with the MicroMount alternative to the traditional crystal mounting loop, the tools now include a wide range of mounts (for both cryocooled and room-temperature crystals), manipulators, and measuring devices. New devices are often available to MacCHESS staff and users for testing.


Latest MicroMount crystal mount, with a thin tip for X-ray
transparency and a thicker base for strength.


Microgripper tool holding a crystal.


Microruler for measuring crystal size.


Tool mounted in a handle.

 

 

Submitted by: Marian Szebenyi, MacCHESS, Cornell University