Knowledge of macromolecular structure is important for understanding and developing treatments for a diverse array of diseases. Most macromolecular structures are determined with X-ray crystallography, for which a key step is to cryogenically cool the crystals to ~100 K in order to mitigate crystal damage from the ionizing radiation. For successful cooling, the crystals are usually pretreated (cryoprotected) using methods that are mainly trial and error and often require extensive screening. Recent work shows that cryoprotection may be optimized using the thermal contraction of the cryosolution as a guiding physical parameter. However, current practice for cryomounting can introduce artifacts from dehydration that complicates thermal contraction based cryoprotection optimization. Further, the thermal contraction is known only for a few simple binary cryosolutions, limiting its general use. Here we propose experiments and technical developments that will not only facilitate straightforward adoption of thermal contraction based cryoprotection optimization, but also provide additional tools for robust handling of fragile macromolecular crystals. A simple and compact apparatus will be developed to maintain humidity during crystal handling and cryomounting, allowing thermal contraction effects rather than dehydration to dominate unit cell shrinkage during execution of cryocooling protocols. A comprehensive study will be conducted to elucidate general principles of the effects of solution composition on thermal contraction. Approaches for vapor diffusion delivery of cryoprotective agents will be further developed. And the possibility of using the diffraction pattern to diagnose suboptimal cryocooling in the context of the thermal contraction model will be explored. The long term effects of the widespread adoption of deductive approaches to cryoprotection optimization on the field of crystallography would be to ensure that the highest quality diffraction possible is recorded from each crystal. This would (a) improve the average quality of structures determined by X-ray crystallography and (b) make the more difficult crystallographic problems more tractable. This work will impact public health by improving the reliability of biological interpretations based on macromolecular structures, and will increase the likelihood that the structure of any particular molecule with a potential impact on health can be efficiently determined. In the long term, this would improve our understanding of the causes of, and facilitate treatments for, diseases resulting from the alteration of macromolecular structure and function by either genetic or environmental factors.
This project will improve the quality of three-dimensional structures of macromolecules determined with macromolecular cryocrystallography. These structures are key for understanding the molecular basis of disease and higher quality structures will in the long run provide more accurate information for developing therapies to improve human health.
| Farley, Christopher; Juers, Douglas H (2014) Efficient cryoprotection of macromolecular crystals using vapor diffusion of volatile alcohols. J Struct Biol 188:102-6 |
| Farley, Christopher; Burks, Geoffry; Siegert, Thomas et al. (2014) Improved reproducibility of unit-cell parameters in macromolecular cryocrystallography by limiting dehydration during crystal mounting. Acta Crystallogr D Biol Crystallogr 70:2111-24 |
| Juers, Douglas H; Ruffin, Jon (2014) MAP_CHANNELS: a computation tool to aid in the visualization and characterization of solvent channels in macromolecular crystals. J Appl Crystallogr 47:2105-2108 |
| Marshall, Haley; Venkat, Murugappan; Seng, Nang San Hti Lar et al. (2012) The use of trimethylamine N-oxide as a primary precipitating agent and related methylamine osmolytes as cryoprotective agents for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr 68:69-81 |
