A high-pressure cryocooling method for protein crystals and biological samples with reduced background X-ray scatter

Abstract

High-pressure cryocooling has been developed as an alternative method for cryopreservation of macromolecular crystals and successfully applied for various technical and scientific studies. The method requires the preservation of crystal hydration as the crystal is pressurized with dry helium gas. Previously, crystal hydration was maintained either by coating crystals with a mineral oil or by enclosing crystals in a capillary which was filled with crystallization mother liquor. These methods are not well suited to weakly diffracting crystals because of the relatively high background scattering from the hydrating materials. Here, an alternative method of crystal hydration, called capillary shielding, is described. The specimen is kept hydrated via vapor diffusion in a shielding capillary while it is being pressure cryocooled. After cryocooling, the shielding capillary is removed to reduce background X-ray scattering. It is shown that, compared to previous crystal-hydration methods, the new hydration method produces superior crystal diffraction with little sign of crystal damage. Using the new method, a weakly diffracting protein crystal may be properly pressure cryocooled with little or no addition of external cryoprotectants, and significantly reduced background scattering can be observed from the resulting sample. Beyond the applications for macromolecular crystallography, it is shown that the method has great potential for the preparation of noncrystalline hydrated biological samples for coherent diffraction imaging with future X-ray sources.

Graphene as a protein crystal mounting material to reduce background scatter

“…This agrees with expectations from a material only ten carbon atoms thick. The lifetimes of the graphene-wrapped cooled and roomtemperature crystals were equivalent to those of crystals obtained using existing methods (Hope, 1988;Berger et al, 2010;Kim et al, 2013). There was no visual evidence of any effect from the physical contact of the graphene and the crystals.…”

“…Flash-cooled Flash-cooled in oil P4 1 2 1 2 P4 1 2 1 2 P4 1 2 1 2 P4 1 2 1 2 P4 1 2 1 2 Unit-cell parameters ( (Kim et al, 2005(Kim et al, , 2013. The graphenewrapping technique gives reproducible results for thaumatin crystals.…”

“…Major sources of background scatter are the mounting materials that typically surround the crystal, such as external mother liquor, coating oil and enclosing capillaries (Kim et al, 2013). Most of these materials are used to hold the crystal in the beam while staving off dehydration and/or protecting the crystal from the damaging effects of ice formation during cryocooling (Garman, 1999(Garman, , 2003Kwong & Liu, 1999;Riboldi-Tunnicliffe & Hilgenfeld, 1999;Garman & Doublié, 2003;Owen et al, 2006;Haas & Rossmann, 1970;Hope, 1988;Henderson, 1990;Juers & Matthews, 2004).…”

Glass-to-cryogenic-liquid transitions in aqueous solutions suggested by crack healing

“…This result suggests that the intermediate states during the phase transition can be expressed as a coexistence of high-density and low-density states, consistent with a first-order phase transition (29)(30)(31)(34)(35)(36)(37). Note that the small-angle X-ray scattering (SAXS) region (Q = 0.3-0.7 Å −1 ) in the WDD profiles is initially low in the HDA state and gradually rises during the phase transition, indicating the structural homogeneity of HDA ice (38), increased density fluctuations during the conversion to LDA, and the structural inhomogeneity of LDA ice (21,22,28). This is consistent with an interpretation in which the low-density state emerges within a high-density matrix during the HDA to LDA transition (1).…”

Practical macromolecular cryocrystallography

“…The oil coating to keep a crystal from dehydrating in the early steps of the procedure may be a disadvantage, since it creates a higher X-ray background, especially for small crystals, and also some mother liquors are soluble in oil. This issue was addressed by using a removable shielding capillary (Kim et al, 2013).…”