Slow cooling of protein crystals

Abstract

Cryoprotectant-free thaumatin crystals have been cooled from 300 to 100 K at a rate of 0.1 K s À1 -10 3 -10 4 times slower than in conventional flash coolingwhile continuously collecting X-ray diffraction data, so as to follow the evolution of protein lattice and solvent properties during cooling. Diffraction patterns show no evidence of crystalline ice at any temperature. This indicates that the lattice of protein molecules is itself an excellent cryoprotectant, and with sodium potassium tartrate incorporated from the 1.5 M mother liquor ice nucleation rates are at least as low as in a 70% glycerol solution. Crystal quality during slow cooling remains high, with an average mosaicity at 100 K of 0.2 . Most of the mosaicity increase occurs above $200 K, where the solvent is still liquid, and is concurrent with an anisotropic contraction of the unit cell. Near 180 K a crossover to solid-like solvent behavior occurs, and on further cooling there is no additional degradation of crystal order. The variation of B factor with temperature shows clear evidence of a protein dynamical transition near 210 K, and at lower temperatures the slope dB/dT is a factor of 3-6 smaller than has been reported for any other protein. These results establish the feasibility of fully temperature controlled studies of protein structure and dynamics between 300 and 100 K.

Glass transition in thaumatin crystals revealed through temperature-dependent radiation-sensitivity measurements

“…However, these temperatures are of the greatest interest for understanding radiation-damage mechanisms because near T = 200 K the protein and solvent system undergoes a dynamical/glass transition (Parak et al, 1982;Doster et al, 1989;Weik et al, 2001Weik et al, , 2004Weik et al, , 2005Gabel et al, 2002). The present study was made possible by the methods and observations described in detail by Warkentin & Thorne (2009), which were in turn inspired by earlier investigations (Weik et al, 2001(Weik et al, , 2005Juers & Matthews, 2001Kriminski et al, 2002;Warkentin et al, 2006). Ice nucleation within protein crystals is strongly suppressed because the solvent is confined within a nanoporous network (Rault et al, 2003) and because much of the solvent is involved in hydrogen-bonding interactions with the protein.…”

“…10 ml drops prepared by mixing 5 ml each of protein and well solution were suspended over 500 ml well solution. No penetrating cryoprotectants were added to the crystals at any temperature and a thorough wash in oil (as described in Warkentin & Thorne, 2009) was sufficient to obtain satisfactory cryocooling.…”

Temperature-dependent macromolecular X-ray crystallography

“…The possibility of performing slow-cooling experiments (Warkentin & Thorne, 2009) has already been discussed in x4. Comparing protein structures determined during slow cooling and during slow heating after flash-cooling might teach us more about the ensemble of conformational substates trapped in a flash-cooled crystalline protein.…”

“…In those cases, the viscosity of the mother liquor confined in the crystal is already sufficiently high to allow vitrification by flash-cooling. Recently, it has been reported that crystalline ice formation does not occur in thaumatin crystals without penetrating cryoprotectants when research papers the temperature is decreased from 300 to 100 K at a very slow rate (0.1 K s À1 ; Warkentin & Thorne, 2009). The potential interest of this new slow-cooling procedure in kinetic crystallography is discussed in x4.…”

“…Movements of side chains and water molecules are then quenched at this temperature and the protein structure determined at 100 K effectively represents that at 200 K. A way to address the degree to which protein structures are quenched during flashcooling is to compare them with structures determined after slow cooling. Warkentin and Thorne recently showed that thaumatin crystals with solvent channels of 25-35 Å can be cooled from room temperature to 100 K at 0.1 K s À1 without crystalline ice formation (Warkentin & Thorne, 2009). An interesting experiment would be to determine the structures of flash-cooled and slow-cooled thaumatin crystals at various temperatures between 100 and 300 K and to compare them.…”