Thorne Group Research and Education

Department of Physics

Laboratory of Atomic and Solid State Physics

Cornell UniversityIthaca, NY • 14853

ret6@cornell.edu • (607)255-6487

New Approaches to Cryo- and Variable Temperature Protein Crystallography

Nearly all protein structures are determined using crystals cooled to T=100 K. However, the cooling process disorders the crystals, and the T=100 K structure may differ in important ways from the biologically active form.

Character and origin of cooling induced disorder in protein crystals

We have used X-ray diffraction imaging (X-ray topography) together with high-resolution mosaicity and q scans to assess cooling-induced disorder in protein crystals. Flash cooled crystals exhibit broadening in both mosiac and q (lattice spacing) scans, and develop a mosaic domain structure. Since water expands on cooling while the protein lattice contracts, this disorder may arise in part from differential thermal expansion of the protein lattice and the solvent within it.

Flash Cooling and Annealing of Protein Crystals. S. Kriminski, C. L. Caylor, M. C. Nonato, K. D. Finkelstein and R. E. Thorne, Acta Cryst. D58, 459-471 (2002).

Heat transfer from protein crystals

We have analyzed heat transfer from protein crystals during cooling in cold gas streams and during plunging into liquid cryogens, and determined the scaling of cooling rates and internal temperature gradients with several relevant experimental parameters. This analysis allows maximum feasible cooling rates as well as maximum crystal temperature rises due to X-ray absorption to be estimated.

Heat Transfer from Protein Crystals: Implications for Flash Cooling and X-ray Beam Heating. S. Kriminski, M. Kazmierczak and R. E. Thorne, Acta Crystallographica D 59,697-708 (2003).

Ultra-Fast and Slow Cooling

Protein conformation is temperature dependent, and so cooling crystals leads to changes in structure. Protein crystals typically contain between 30 and 90% water. On cooling, this water may crystallize, producing ice diffraction that interferes with that from the protein, and damaging the protein lattice. One might guess that there are two favorable limits for cooling crystals from room temperature to T=100 K:

However, cooling rates achieved in standard practice are in neither of these favorable limits.

Ultra-Fast Cooling:

The fastest cooling rates are obtained by plunging samples from air into a cryogenic liquid such as nitrogen, propane or ethane. We have demonstrated a simple plunge cooling method that increases cooling rates for small samples by a factor of 20-100 over previous best practice. These cooling rates - 20,000 to 100,000 K/s - allow even protein-free aqueous solutions to be vitrified with very small (<10%) cryoprotectant concentrations.

Above any cold liquid surface there will be a layer of cold gas, formed by conduction and convection and, if the cryogenic liquid is near its boiling point, by evaporation. For sufficiently small samples, most cooling occurs in this gas layer, not in the liquid, at a rate determined by heat transfer to the gas. Our measurements showed that "sufficiently small" is smaller than roughly 500 microns - which is larger than nearly all protein crystals. By simply blowing this cold gas layer away immediately prior to the sample plunge, we increased cooling rates from ~1000 K/s to ~20,000 K/s for ordinary size crystals.

Hyperquenching for Protein Crystallography. M. Warkentin, V. Berejnov, and R. E. Thorne, J. Appl. Cryst. J. Appl. Cryst. 39, 805-811 (2006).

Slow Cooling:

The obvious obstacle to successful slow cooling is ice crystal nucleation and growth. It has long been assumed - and observed - that protein crystals must be rapidly cooled to prevent ice formation. We have shown that protein crystals with solvent contents up to at least 65% can be cooled to T=100 K at only 0.1 K/s without appreciable ice formation, often without any penetrating cryoprotectants. This remarkable result indicates that ice nucleation is strongly suppressed by confinement in the nanoporous network found inside protein crystals, and that nearly all ice seen in diffraction patterns nucleates in the solvent outside the crystal.

Slow cooling of protein crystals. M. Warkentin and R. E. Thorne, J. Appl. Cryst. 42 944-952 (2009).

Cryocrystallography without cryoprotectants

Protein crystallographers have long assumed that penetrating cryoprotectants are needed to prevent ice formation inside their crystals, and much time and effort is expended finding suitable cryoprotectants and concentrations.

We have shown that essentially all crystals can be successfully cooled to T=100 K without any penetrating cryoprotectants using our hyperquenching method, provided that the external solvent is removed, e.g., using oil, blotting or dehydration.

A general method for hyperquenching protein crystals. M. Warkentin and R. E. Thorne, J. Struct. Funct. Gen. 8, 141-144 (2007).

For many protein crystals (with solvent contents up to at least 65%), no penetrating cryoprotectants are necessary even when crystals are cooled very slowly, provided all external solvent is removed.

Slow cooling and temperature-controlled protein crystallography. M. Warkentin and R. E. Thorne, J. Struct. Funct. Gen. 11, 85-89 (2010).

Variable temperature structure determination

In the more than 60 year history of protein crystallography, the structures of only a handful of proteins have been determined at temperatures other than 300 and 100 K. The temperature evolution of structure - especially between T=300 and 180 K - provides insight into conformational energy landscapes, the stability of protein folds, and possible mechanisms of enzymatic action. The obstacle to such studies has been ice formation, which has prevented structure determination between ~240 and ~180 K.

Using insights from our slow cooling studies, we have developed methods for full variable temperature data collection at all temperatures between T=300 K and 100 K. We have used these methods to study structure evolution near the active site in the enzyme urease, and to understand them mechanisms of radiation damage to protein crystals.

Glass transition in thaumatin crystals revealed through temperature dependent radiation sensitivity measurements. M. Warkentin and R. E. Thorne, Acta Cryst. D 66, 1092-1100 (2010).

Dark progression reveals slow timescales for radiation damage between T=180 and 240 K. M. Warkentin, R. Badeau, J. Hopkins and R. E. Thorne, Acta Cryst. D 67, 792-803 (2011).

Spatial distribution of radiation damage to crystalline proteins at 25 to 300 K. M. Warkentin, R. Badeau, J. B. Hopkins and R. E. Thorne, Acta Cryst. D68, 1108-1117 (2012).

Kinetic trapping of biologically relevant conformations

We have combined our hyperquenching and slow-cooling methods to study how T=100 K protein conformations depend upon the cooling rate. In the case of urease, unlike conventional plunge cooling, hyperquenching traps the room-temperature conformation. The potential impact of this approach on structural studies of proteins is large.

New crystal mounting methods for cryo- and room-temperature crystallography

We developed microfabricated ultra-X-ray transparent tools for crystal harvesting and X-ray data collection, especially for harvesting and data collection from microcrystals. These tools have been patented and commercialized by MiTeGen, LLC, and are now used in small molecule and protein crystallography laboratories around the world.

Microfabricated Mounts for Microcrystal Cryocrystallography. R. E. Thorne, Z. Stum, J. Kmetko and K. O'Neill, J. Appl. Cryst. 36, 1455-1460 (2003).

Part of the reason why room-temperature X-ray data collection has all but been abandoned is the difficulty of mounting and handling crystals in convention X-ray capillaries. We have developed a new method for room-temperature sample mounting that allows crystals to be mounted in seconds with essentially 100% chance of success, and to be shipped to the synchrotron for data collection. This mounting method has been patented, and has been commercialized by MiTeGen, LLC.

A new sample mounting technique for room-temperature macromolecular crystallography. Y. Kalinin, J. Kmetko, A. Bartnik, A. Stewart, R. Gillilan, E. Lobkovsky and R. E. Thorne, J. Appl. Cryst. 38, 333-339 (2005).